Modification of the tumor microenvironment enhances immunity with plasmid gene therapy.

Local intratumor delivery with electroporation of low levels of plasmids encoding molecules, induces an antitumor effect without causing systemic toxicity. However, previous studies have predominately focused on the function of the delivered molecule encoded within the plasmid, and ignored the plasmid vector. In this study, we found vectors pUMVC3 and pVax1 induced upregulation of MHC class I (MHC-I) and PD-L1 on tumor cell surface. These molecules participate in a considerable number of immunoregulatory functions through their interactions with and activating inhibitory immune cell receptors. MHC molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells and tumor cells. Increased PD-L1 expression on tumor cells is an important monitor of tumor growth and the effectiveness of immune inhibitor therapy. Results from flow cytometry confirmed increased expression of MHC-I and PDL-1 on B16F10, 4T1, and KPC tumor cell lines. Preliminary animal data from tumor-bearing models, B16F10 melanoma, 4T1 breast cancer and KPC pancreatic cancer mouse models showed that tumor growth was attenuated after pUMVC3 intratumoral electroporation. Our data also documented that pSTAT1 signaling pathway might not be associated with plasmid vectors' function of upregulating MHC-I, PD-L1 on tumor cells.

IL-12 and PD-1 peptide combination gene therapy for the treatment of melanoma.

Interleukin-12 (IL-12) gene electrotransfer (GET) delivery is highly effective in inducing long-term, complete regression in mouse and human melanoma and other solid tumors. Therapeutic efficacy is enhanced by immune checkpoint inhibitors, and the combination of IL-12 plasmid GET (pIL-12 GET) and anti-programmed cell death protein 1 (PD-1) monoclonal antibodies has reached clinical trials. In this study, we designed peptides and plasmids encoding the mouse homologs of the pembrolizumab and nivolumab programmed cell death 1 ligand 1 (PD-L1) binding regions. We hypothesized that intratumor autocrine/paracrine peptide expression would block PD-1/PD-L1 binding and provide cancer patients with an effective and cost-efficient treatment alternative. We demonstrated that the mouse homolog to pembrolizumab was effective at blocking PD-1/PD-L1 in vitro. After intratumor plasmid delivery, both peptides bound PD-L1 on tumor cells. We established that plasmid DNA delivery to tumors in vivo or to tumor cells in vitro upregulated several immune modulators and PD-L1 mRNA and protein, potentiating this therapy. Finally, we tested the combination of pIL-12 GET therapy and peptide plasmids. We determined that pIL-12 GET therapeutic efficacy could be enhanced by combination with the plasmid encoding the pembrolizumab mouse homolog.

[Construction of a new oncolytic virus oHSV2hGM-CSF and its anti-tumor effects].

OBJECTIVE: The aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF. METHODS: oHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded. RESULTS: Both oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P < 0.01). The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree. CONCLUSION: The findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vive to the transplanted B16R tumor models.

[Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP (HSV(GFP))].

OBJECTIVE: To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)). METHODS: Tumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. RESULTS: HSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol. CONCLUSION: Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

[Generation of a herpes simplex virus-permissive mouse melanoma cell line B16RHSV].

OBJECTIVE: To generate an oncolytic herpes simplex virus (oHSV) permissive mouse melanoma cell line B16RHSV, preserving the tumorigenic ability in syngeneic mice. METHODS: The herpes simplex virus entry mediator (HVEM) gene was amplified by PCR from human melanoma cell line A375, and cloned into pGEM-T Easy vector for sequencing. The HVEM gene was then cloned into pcDNA3 vector to generate pcDNA3-HVEM for transfection of mouse melanoma cell line B16-F10 cells. After that, the putative transfected cells were selected in full growth medium containing G418. The HVEM-expressing cells were isolated by immunomagnetic bead separation. The mouse melanoma cell line expressing oHSV receptor-HVEM, designated as B16RHSV, was generated. The permissibility of B16RHSV cells to oHSV infection was examined with green fluorescence protein (GFP)-expressing oHSV (oHSVGFP). To investigate the tumorigenic ability of both cells in vivo, 2×10(5) cells in 100 µl were subcutaneously inoculated into the right flanks of C57/BL mice. RESULTS: In vitro, the B16RHSV mouse melanoma cells were shown by fluorescence microscopy capable of being infected by oHSVGFP. In vivo, the B16RHSV cells, like their wild type counterpart, grew to form melanoma in syngeneic mice. CONCLUSION: A herpes simplex virus-permissive mouse melanoma cell line was established. Its tumorigenicity remained unchanged.

Modification of the Tumor Microenvironment Enhances Anti-PD-1 Immunotherapy in Metastatic Melanoma.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. Both primary and acquired resistance have become major obstacles, greatly limiting the long-lasting effects and wide application of blockade therapy. Many patients with metastatic melanoma eventually require further therapy. The absence of T-cell infiltration to the tumor site is a well-accepted contributor limiting immune checkpoint inhibitor efficacy. In this study, we combined intratumoral injection of plasmid IL-12 with electrotransfer and anti-PD-1 in metastatic B16F10 melanoma tumor model to increase tumor-infiltrating lymphocytes and improve therapeutic efficacy. We showed that effective anti-tumor responses required a subset of tumor-infiltrating CD8+ and CD4+ T cells. Additionally, the combination therapy induced higher MHC-I surface expression on tumor cells to hamper tumor cells escaping from immune recognition. Furthermore, we found that activating T cells by exposure to IL-12 resulted in tumors sensitized to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.

Determination of Cytotoxic Activities Against Melanoma Cells Using Flow Cytometry.

Within the adaptive and innate immune system, effector lymphocytes known as cytotoxic T cells (CTLs) or natural killer (NK) cells play an essential role in host defense against tumor cells and virally infected cells. Here we describe a flow cytometry-based method to quantify CTLs or NK cell cytotoxic activity against melanoma cells. In this assay, spleen cells, peripheral blood mononuclear cells (PBMCs), or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line. The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells. After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications.

Controlled Delivery of Plasmid DNA to Melanoma Tumors by Gene Electrotransfer.

Gene electrotransfer (GET) is a reliable and effective physical method for in vivo delivery of plasmid DNA (pDNA). Several preclinical and clinical studies have utilized GET to deliver plasmids encoding immune stimulating genes for treatment of melanoma and other tumor types. Intratumor delivery of plasmids encoding cytokines directly to tumors can induce not only a local immune response, but a systemic one as well. To obtain an effective immune response, it is critical to achieve the appropriate expression pattern of the delivered transgene. Expression pattern (levels and kinetics) can be modified by manipulating the electrotransfer parameters. These parameters include the tissue target and the electric pulse parameters of pulse width, electric field, and pulse number. We have found that to induce a robust immune response, we needed only low to moderately elevated expression levels compared to controls. When developing a therapeutic protocol, it is important to establish what expression profile will enable the appropriate response. In this chapter we describe how to determine the appropriate GET protocol to achieve the expression profile that can result in the desired clinical response.

Interferon-gamma inhibits aldehyde dehydrogenasebright cancer stem cells in the 4T1 mouse model of breast cancer.

BACKGROUND: Despite improvements in disease diagnosis, treatment, and prognosis, breast cancer is still a leading cause of cancer death for women. Compelling evidence suggests that targeting cancer stem cells (CSCs) have a crucial impact on overcoming the current shortcomings of chemotherapy and radiotherapy. In the present study, we aimed to study the effects of T cells and a critical anti-tumor cytokine, interferon-gamma (IFN-γ), on breast cancer stem cells. METHODS: BALB/c mice and BALB/c nude mice were subcutaneously injected with 4T1 tumor cells. Tumor growth and pulmonary metastasis were assessed. ALDEFLOUR™ assays were performed to identify aldehyde dehydrogenasebright (ALDHbr) tumor cells. ALDHbr cells as well as T cells from tumor-bearing BALB/c mice were analyzed using flow cytometry. The effects of CD8+ T cells on ALDHbr tumor cells were assessed in vitro and in vivo. The expression profiles of ALDHbr and ALDHdim 4T1 tumor cells were determined. The levels of plasma IFN-γ were measured by enzyme-linked immunosorbent assay, and their associations with the percentages of ALDHbr tumor cells were evaluated. The effects of IFN-γ on ALDH expression and the malignancy of 4T1 tumor cells were analyzed in vitro. RESULTS: There were fewer metastatic nodules in tumor-bearing BALB/c mice than those in tumor-bearing BALB/c nude mice (25.40 vs. 54.67, P < 0.050). CD8+ T cells decreased the percentages of ALDHbr 4T1 tumor cells in vitro (control vs. effector to target ratio of 1:1, 10.15% vs. 5.76%, P < 0.050) and in vivo (control vs. CD8+ T cell depletion, 10.15% vs. 21.75%, P < 0.001). The functions of upregulated genes in ALDHbr 4T1 tumor cells were enriched in the pathway of response to IFN-γ. The levels of plasma IFN-γ decreased gradually in tumor-bearing BALB/c mice, while the percentages of ALDHbr tumor cells in primary tumors increased. IFN-γ at a concentration of 26.68 ng/mL decreased the percentages of ALDHbr 4T1 tumor cells (22.88% vs. 9.88%, P < 0.050) and the protein levels of aldehyde dehydrogenase 1 family member A1 in 4T1 tumor cells (0.86 vs. 0.49, P < 0.050) and inhibited the abilities of sphere formation (sphere diameter <200 µm, 159.50 vs. 72.0; ≥200 µm, 127.0 vs. 59.0; both P < 0.050) and invasion (89.67 vs. 67.67, P < 0.001) of 4T1 tumor cells. CONCLUSION: CD8+ T cells and IFN-γ decreased CSC numbers in a 4T1 mouse model of breast cancer. The application of IFN-γ may be a potential strategy for reducing CSCs in breast cancer.

Myeloid-derived suppressor cells infiltration in non-small-cell lung cancer tumor and MAGE-A4 and NY-ESO-1 expression.

Cancer/testis antigens melanoma-associated antigen 4 (MAGE-A4) and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) are of clinical interest as biomarkers and present valuable targets for immunotherapy; however, they are poor prognostic markers in non-small cell lung cancer (NSCLC). In addition, myeloid derived suppressor cells (MDSCs) are recognized as a key element in tumor escape and progression. The aim of the present study was to investigate the diagnostic and prognostic value of MAGE-A4 and NY-ESO-1, and their association with MDSCs in NSCLC samples. The expression levels of MAGE-A4 and NY-ESO-1, and the infiltration of MDSCs (CD33+), were analyzed by immunohistochemistry of 67 tissue samples from patients with NSCLC. Overall, 58.33% of the NSCLC squamous cell carcinoma tissues and 94.7% of adenocarcinoma tissues were positive for MAGE-A4. NY-ESO-1 expression was observed in 52.78% of the squamous cell carcinoma tissues and 80% of the adenocarcinoma tissues. In primary adenocarcinoma tumor tissues, MAGE-A4 and NY-ESO-1 demonstrated a higher intensity of expression compared with the squamous cell carcinoma tissues. A total of 33 (91.7%) squamous cell carcinoma and 19 (95.0%) adenocarcinoma specimens were positive for CD33. The expression of MAGE-A4 and NY-ESO-1 antigens and infiltration of MDSCs was associated with poor prognosis of patients with NSCLC. Further studies investigating the association between these findings and underlying molecular mechanisms are required.

IL-12 Gene Electrotransfer Triggers a Change in Immune Response within Mouse Tumors.

Metastatic melanoma is an aggressive skin cancer with a relatively low survival rate. Immune-based therapies have shown promise in the treatment of melanoma, but overall complete response rates are still low. Previous studies have demonstrated the potential of plasmid IL-12 (pIL-12) delivered by gene electrotransfer (GET) to be an effective immunotherapy for melanoma. However, events occurring in the tumor microenvironment following delivery have not been delineated. Therefore, utilizing a B16F10 mouse melanoma model, we evaluated changes in the tumor microenvironment following delivery of pIL-12 using different GET parameters or injection of plasmid alone. The results revealed a unique immune cell composition after intratumoral injection of pIL-12 GET. The number of immune memory cells was markedly increased in pIL-12 GET melanoma groups compared to control group. This was validated using flow cytometry to analyze peripheral blood mononuclear cells as well as delineating immune cell content using immunohistochemistry. Significant differences in multiple cell types were observed, including CD8⁺ T cells, regulatory T cells and myeloid cells, which were induced to mount a CD8⁺PD1- T cells immune response. Taken together, these findings suggest a basic understanding of the sequence of immune activity following pIL-12 GET and also illuminates that adjuvant immunotherapy can have a positive influence on the host immune response to cancer.

Antidepressant-like effects of a novel curcumin derivative J147: Involvement of 5-HT1A receptor.

Depression is a dysthymia disorder characterized by a pervasive or persistent mental disorder that causes mood, cognitive and memory deficits. J147, a curcumin analogue, increases brain derived neurotrophic factor (BDNF) levels and facilitates memory in animals. Because curcumin has the antidepressant-like activity, the present study investigated the potential antidepressant-like effects of J147 in the forced swimming test (FST) and tail suspension tests (TST) and the involvement of 5-HT receptors related to cAMP signaling. The results suggested that acute treatment of J147 at doses of 5 and 10 mg/kg via gavage markedly reduced the duration of immobility in both TST and FST, either 1 h or 3 h after treatment, respectively. It did not alter locomotor activity but influence the immobile response. The molecular biological assays showed that 5-HT1A receptor expression was significantly increased at 1 h after treatment with J147 at a dose of 10 mg/kg. In addition, pre-treatment of mice with WAY-100635 blocked the J147's effect in the FST. 5-HT1B receptor expression was not significantly increased with increasing doses of J147. The 5-HT1B receptors antagonist isamoltan partially prevented J147's effect in the FST. The levels of downstream molecular targets, cAMP, PKA, pCREB and BDNF were significantly increased 1 h after treatment with J147 at doses of 5 and 10 mg/kg. The up-regulated pCREB and BDNF levels lasted for 3 h after 10 mg/kg of J147. These findings demonstrated that J147 has antidepressant-like effects that are mediated, at least in part, by activating the 5-HT1A/cAMP/PKA/CREB/BDNF-signaling pathway.

A novel PDE9 inhibitor WYQ-C36D ameliorates corticosterone-induced neurotoxicity and depression-like behaviors by cGMP-CREB-related signaling.

BACKGROUND: Major depressive disorder (MDD) is a mental disease characterized by depressed mood, lifetime anxiety, and deficits of learning and memory. Inhibition of phosphodiesterase 9 (PDE9) has been reported to improve rodent cognitive and memory function. However, the role of PDE9 in MDD, in particular its manifestations of depression and anxiety, has not been investigated. METHODS: We examined the protective effects of WYQ-C36D (C36D), a novel PDE9 inhibitor, against corticosterone-induced cytotoxicity, pCREB/CREB and BDNF expression by cell viability, and immunoblot assays in HT-22 cells. The potential effects of C36D at doses of 0.1, 0.5, and 1 mg/kg on stress-induced depression- and anxiety-like behaviors and memory deficits were also examined in mice. RESULTS: C36D significantly protected HT-22 cells against corticosterone-induced cytotoxicity and rescued corticosterone-induced decreases in cGMP, CREB phosphorylation, and BDNF expression. All these effects were otherwise blocked by the PKG inhibitor Rp-8-Br-PET-cGMPS (Rp8). In addition, when tested in vivo in stressed mice, C36D produced antidepressant-like effects on behavior, as shown by decreased immobility time both in the forced swimming and tail suspension tests. C36D also showed anxiolytic-like and memory-enhancing effects in the elevated plus-maze and novel object recognition tests. CONCLUSION: Our results show that inhibition of PDE9 by C36D produces antidepressant- and anxiolytic-like behavioral effects and memory enhancement by activating cGMP/PKG signaling pathway. PDE9 inhibitors may have the potential as a novel class of drug to treat MDD.

[Effects of jieyuwan on HPA axis and immune system in chronic stress models in rats].

OBJECTIVE: To observe the effects of jieyuwan on neuroendocrine-immune of chronic unpredictable stress rats, for identifing its mechanism of action. METHOD: Animals were randomly assigned to six groups: one non-stressed (control) group, one stressed group (CUS + vehicle) , three treatment groups (CUS + Jieyuwan 53, 106, 212 mg x kg(-1), respectively), and one group for imipramine (CUS + imipramine 10 mg x kg(-1)). We used the chronic unpredictable stress (CUS) test in rats to investigated the effects of chronic oral administration (21 days) of Jieyuwan and imipramine (ip) on the sucrose consumption, the relative weight of adrenal and thymus, the concentration of serum corticosterone, adrenocorticotropic hormone, TNF-alpha, IL-1beta measured by ELISA. RESULT: Our results showed that Sprague-Dawley rats subjected to CUS exhibited increased adrenal weight, reduction in thymus weight, less sucrose consumption, and synchronized with higher serum corticosterone, adrenocorticotropic hormone, TNF-alpha, IL-1beta concentration. Chronic treatment with Jieyuwan (53, 106, 212 mg x kg(-1), po, 21 days) could improve locomotion of stressed rat sucrose consumption, turn over the high levels of serum corticosterone, adrenocorticotropic hormone, TNF-alpha, IL-1beta concentration. CONCLUSION: The therapeutic actions of jieyuwan, in some degree, may be related with indirect or direct effects on HPA axis and immune system.

Dnase1L3 Regulates Inflammasome-Dependent Cytokine Secretion.

Pediatric-onset systemic lupus erythematosus arises in humans and mice lacking the endonuclease Dnase1L3. When Dnase1L3 is absent, DNA from circulating apoptotic bodies is not cleared, leading to anti-DNA antibody production. Compared to early anti-DNA and anti-chromatin responses, other autoantibody responses and general immune activation in Dnase1L3-/- mice are greatly delayed. We investigated the possibility that immune activation, specifically inflammasome activation, is regulated by Dnase1L3. Here, we report that Dnase1L3 inhibition blocked both NLR family, pyrin domain containing 3 (NLRP3) and NLRC4 inflammasome-mediated release of high-mobility group box 1 protein and IL-1ß. In contrast to IL-1ß release, Dnase1L3 inhibition only mildly impaired NLRP3-dependent pyroptosis, as measured by propidium iodide uptake or LDH release. Mechanistically, we found that Dnase1L3 was needed to promote apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) nuclear export and speck formation. Our results demonstrate that Dnase1L3 inhibition separates cytokine secretion from pyroptosis by targeting ASC. These findings suggest that Dnase1L3 is necessary for cytokine secretion following inflammasome activation.

Intrinsic repair protects cells from pore-forming toxins by microvesicle shedding.

Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term 'intrinsic repair', and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.

Myeloid-derived suppressor cells enhance the expression of melanoma-associated antigen A4 in a Lewis lung cancer murine model.

The cancer-testis (CT) family of antigens are expressed in multiple types of malignant neoplasm and are silent in normal tissues, apart from the testis. Immunotherapy targeting CT antigens is a promising therapeutic strategy for treatment of solid tumors. One member of this family, melanoma-associated antigen A4 (MAGE-A4), has been demonstrated to be expressed in melanomas and lung cancer. Patients with tumors expressing the MAGE-A4 antigen exhibit specific cellular and humoral immune responses to the antigen, resulting in a favorable prognosis. Conversely, the expression of MAGE-A4 is associated with poor survival in lung cancer. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immunosuppressive cells, which are upregulated in the cancer microenvironment. Little is known regarding any potential correlation between the expression of MAGE-A4 antigens and the accumulation of MDSCs. The present study aimed to examine the association between circulating MDSC levels and MAGE-A4 expression in a mouse model of Lewis lung cancer. The expression of MAGE-A4 in tumor cells or tissues was evaluated using western blotting, while the percentage of MDSCs (CD11b+Gr-1+) in the blood was detected by flow cytometry. In addition, the suppressive capacity of MDSCs and the effectiveness of MDSC depletion were assessed in C57BL/6 tumor-bearing mice. MDSCs were demonstrated to upregulate MAGE-A4 expression via the phosphosphorylated-signal transducer and activator of transcription 3705 pathway, while depletion of MDSCs decreased the tumor growth rate, prolonged median survival and enhanced the recognition of MAGE-A4 by CD8+ T cells. These findings indicated that immunotherapeutic strategies involving induction of cytotoxic T lymphocytes that target MAGE-A4, in combination with MDSC depletion, may be an effective approach to immunotherapy for cancer types with high expression of MAGE-A4.

LAPTM4B-35 protein is a weak tumor-associated antigen candidate.

Lysosome-associated protein transmembrane 4ß (LAPTM4B) is a gene that has been indicated to be involved in cancer. It is located at chromosome 8q22 and is composed of seven exons and six introns. LAPTM4B encodes two protein isoforms: LAPTM4B-35 and LAPTM4B-24. LAPTM4B-35 is markedly upregulated and LAPTM4B-24 is downregulated in several types of cancer. LAPTM4B-35 is 91 amino acids (N91) longer than LAPTM4B-24 at the N-terminus. In the present study, western blotting, enzyme-linked immunosorbent spot analysis and the B16F10-N91 tumor bearing-mice experiments were used to evaluate whether the overexpression of N91 indicates its potential as a candidate tumor-associated antigen. The results revealed that N91 was expressed in a wide range of normal mouse tissues and human peripheral blood mononuclear cells, with varying expression levels. The weak immunogenicity of N91 protein suggested it was a weak candidate antigen; however, the N91 protein was associated with cell proliferation.

Antitumor enhancement by adoptive transfer of tumor antigen primed, inactivated MHC-haploidentical lymphocytes.

The present study investigated the antitumor effects by adoptive transfer of tumor antigen primed, inactivated MHC-haploidentical lymphocytes in TC-1 lung cancer mouse model. Our studies revealed that the inactivated MHC-haploidentical effecter cells display the antitumor activity in vitro and target the tumor in vivo. After adoptive transferring these effecter cells, the Th1 cytokines such as IL-2 and IFN-γ are elevated in the serum; the recipient tumor-specific cytotoxic T-cells and natural killer cells are activated; tumor specific memory T cells are induced; tumor growth is inhibited and mouse survival is prolonged. The results indicate that MHC-haploidentical lymphocytes provide both effecter cells which can target the tumor cells through the identical MHC molecules and an adjuvant effects through the unmatched allogeneic MHC molecules which induces endogenous innate and adaptive antitumor immune responses.