Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model.
ABSTRACT: BACKGROUND: The novel cytokine, interleukin (IL)-18, is a strong interferon-gamma inducer and costimulatory factor in Th1 cell activation. IL-18 triggers IFN-gamma production and enhances cytolytic activity in both T and NK cells. However, the exact mechanism of antitumor action of IL-18 remains to be clarified. To determine the effects of IL-18 plasmid DNA on hepatic cancer in mice, CT26 murine colon adenocarcinoma cells were established in mouse liver. METHODS: Plasmid vectors encoding IL-18 were transferred directly into the liver 7 days after tumor injection to restrict IL-18 expression within the tumor site. The IL-18 protein level was increased in the liver 4 days after plasmid injection, and a marked antitumoral effect was observed at day 7. Antitumor effects were evaluated by measuring tumor regression, immune cell population, and IFN-gamma production. RESULTS: The IL-18 plasmid controlled the growth of hepatic tumors and proliferation of splenic immune cells. Moreover, treatment of CT26 tumors with the IL-18 plasmid significantly enhanced the population of the effector T and NK cells in the spleen and peripheral blood. In spleen, the population of CD4+CD62Low cells was augmented in response to IL-18 on day 7. These results are consistent with the increase in CD4+ T cells secreting IFN-gamma, but not CD8+ T cells. The marked reduction of tumor growth in tumor-bearing mice was associated with the maintenance of IFN-gamma production in spleen in response to IL-18. These antitumoral effects were maintained until 14 days after plasmid injection. CONCLUSION: Our results suggest that direct plasmid DNA transfer of IL-18 with no accompanying reagents to augment transfection efficiency may be useful in tumor immunotherapy.
Project description:We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11. The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) production in T cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM-CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-gamma did not. In cocultures with osteoblasts and spleen cells from IFN-gamma receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-gamma production: IFN-gamma had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were from wild-type mice and vice versa, we identified that the target cells for IFN-gamma inhibition of OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-gamma production.
Project description:Studies of tumor models using syngeneic transplantation have advanced our understanding of tumor immunity, including both immune surveillance and evasion. Murine mammary carcinoma 4T1 cells secrete immunosuppressive soluble factors as demonstrated in splenocyte culture. Cultured primary splenocytes secrete IFN-γ, which was strikingly elevated when the cells were isolated from 4T1 tumor-bearing mice. The secretion of IFN-γ peaked a week after 4T1 inoculation and then declined. This reduction may be due to the relatively decreased lymphocytes and increased granulocytes in a spleen accompanied by splenomegaly with time after the 4T1 inoculation. IFN-γ production was further suppressed with the addition of the conditioned media from 4T1 cells to the splenocyte culture. This suppressive effect was more evident in the splenocytes isolated from mice that had 4T1 tumors for a longer period of time and was not observed in the conditioned medium either from CT26 cells or with splenocytes isolated from CT26 tumor-bearing mice. These results suggest that the IFN-γ suppression is 4T1 tumor-specific. The soluble factor(s) in the 4T1-conditioned media was a protein between 10 to 100 kDa. The cytokine tip assay demonstrated several known cytokines that negatively regulate immune responses and may be candidates for this immunosuppression activity.
Project description:Interferon (IFN)-gamma plays an important role in the innate immune response against intracellular bacterial pathogens. It is commonly thought that natural killer cells are the primary source of this cytokine that is involved in activating antibacterial effects in infected cells and polarizing CD4+ T cells toward the Th1 subset. However, here we show that both effector and memory CD8+ T cells have the potential to secrete IFN-gamma in response to interleukin (IL)-12 and IL-18 in the absence of cognate antigen. We demonstrate that memory CD8+ T cells specific for the ovalbumin protein secrete IFN-gamma rapidly after infection with wild-type Listeria monocytogenes (LM). Furthermore, small numbers of ovalbumin-specific, memory CD8+ T cells can reduce spleen and liver bacterial counts in IFN-gamma-deficient mice 3 d after LM infection. Up-regulation of the receptors for IL-12 and IL-18 provides a mechanism for the ability of memory CD8+ T cells to respond in this antigen nonspecific manner. Thus, CD8+ T cells play an important role in the innate immune response against intracellular pathogens by rapidly secreting IFN-gamma in response to IL-12 and IL-18.
Project description:Interleukin-18 (IL-18) is a proinflammatory cytokine that promotes natural killer (NK) and T-cell activation. Several poxviruses, including vaccinia virus (VV), encode a soluble IL-18-binding protein (IL-18bp). The role of the VV IL-18bp (gene C12L) in vivo was studied with wild-type (vC12L), deletion mutant (vDeltaC12L), and revertant (vC12L-rev) viruses in a murine intranasal model of infection. The data show that vDeltaC12L was markedly attenuated, characterized by a mild weight loss and reduced virus titers in lungs, brain, and spleen. Three days after infection, NK cytotoxic activity was augmented in the lung, spleen, and mediastinal lymph nodes (MLNs) of vDeltaC12L-infected mice compared to controls. Seven days after infection, vDeltaC12L-infected mice displayed heightened VV-specific cytotoxic T-lymphocyte (CTL) responses in the lungs, spleen, and MLNs. Gamma interferon (IFN-gamma) levels were also dramatically elevated in lavage fluids and cells from lungs of mice infected with vDeltaC12L. Finally, we demonstrate that IL-18 is produced in vitro and in vivo after VV infection. Taken together, these data demonstrate a role for the vIL-18bp in counteracting IL-18 in both the innate and the specific immune response to VV infection and indicate that the ability of IL-18 to promote vigorous T-cell responses (cytotoxic activity and IFN-gamma production) is a critical factor in the accelerated clearance of the vDeltaC12L mutant.
Project description:BACKGROUND:Although the whole tumor cell vaccine can provide the best source of immunizing antigens, there is still a limitation that most tumors are not naturally immunogenic. Tumor cells genetically modified to secrete immune activating cytokines have been proved to be more immunogenic. IL-18 could augment proliferation of T cells and cytotoxicity of NK cells. GM-CSF could stimulate dendritic cells, macrophages and enhance presentation of tumor antigens. In our study, we used mouse GM-CSF combined with IL-18 to modify Lewis lung cancer LL/2, then investigated whether vaccination could suppress tumor growth and promote survival. METHODS:The Lewis lung cancer LL/2 was transfected with co-expressing mouse GM-CSF and IL-18 plasmid by cationic liposome, then irradiated with a sublethal dose X ray (100 Gy) to prepare vaccines. Mice were subcutaneously immunized with this inactivated vaccine and then inoculated with autologous LL/2 to estimate the antitumor efficacy. RESULTS:The studies reported here showed that LL/2 tumor cell vaccine modified by a co-expressing mouse GM-CSF and IL-18 plasmid could significantly inhibit tumor growth and increased survival of the mice bearing LL/2 tumor whether prophylactic or adoptive immunotherapy in vivo. A significant reduction of proliferation and increase of apoptosis were also observed in the tumor treated with vaccine of co-expressing GM-CSF and IL-18. The potent antitumor effect correlated with higher secretion levels of pro-inflammatory cytokines such as IL-18, GM-CSF, interferon-? in serum, the proliferation of CD4+ IFN-?+, CD8+ IFN-?+ T lymphocytes in spleen and the infiltration of CD4+, CD8+ T in tumor. Furthermore, the mechanism of tumor-specific immune response was further proved by 51Cr cytotoxicity assay in vitro and depletion of CD4, CD8, NK immune cell subsets in vivo. The results suggested that the antitumor mechanism was mainly depended on CD4+, CD8+ T lymphocytes. CONCLUSIONS:These results provide a new insight into therapeutic mechanisms of IL-18 plus GM-CSF modified tumor cell vaccine and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.
Project description:Monokines (i.e., interleukin [IL]-12, -18, and -15) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma), which is a critical factor for immune surveillance of cancer and monocyte clearance of infection. We show that SET, which is a potent inhibitor of protein phosphatase type 2A (PP2A) activity, is highly expressed in human CD56bright NK cells, which produce more IFN-gamma than CD56dim NK cells. SET was up-regulated upon monokine stimulation of primary human NK cells. Furthermore, ectopic overexpression of SET significantly enhanced IFN-gamma gene expression in monokine-stimulated NK cells. In contrast, RNAi-mediated suppression of SET expression renders NK cells inefficient in producing high levels of IFN-gamma in response to monokine costimulation. Mechanistically, suppression of PP2A activity by SET is important for IFN-gamma gene expression in NK cells. In fact, treatment of primary human NK cells with the PP2A activator 1,9-dideoxy-forskolin, as well as administration of the drug to C57BL/6 mice, significantly reduced NK-dependent IFN-gamma production in response to monokine treatment. Further, SET knockdown or pharmacologic activation of PP2A diminished extracellular signal-regulated kinase 1/2, p65RelA, signal transducer and activator of transduction 4 (STAT4), and STAT5 activity in monokine-stimulated NK cells, potentially contributing to the reduction in IFN-gamma gene expression. Thus, SET expression is essential for suppressing PP2A phosphatase activity that would otherwise limit NK cell antitumoral and/or antiinflammatory functions by impairing NK cell production of IFN-gamma.
Project description:Recent developments in therapeutic strategies have improved the prognosis of head and neck squamous cell carcinoma (HNSCC). Nevertheless, 5-year survival rate remains only 40%, necessitating new therapeutic agents. Oncolytic virotherapy entails use of replication-competent viruses to selectively kill cancer cells. We aimed to explore the potential of HF10 as an oncolytic virus against human or mouse HNSCC cell lines, and primary-cultured HNSCC cells. HF10 replicated well in all the HNSCC cells, in which it induced cytopathic effects and cell killing. Next, we investigated the oncolytic effects of HF10 in ear tumor models with human or mouse tumor cells. We detected HF10-infected cells within the ear tumors based on their expression of green fluorescent protein. HF10 injection suppressed ear tumor growth and prolonged overall survival. In the syngeneic model, HF10 infection induced tumor necrosis with infiltration of CD8-positive cells. Moreover, the splenocytes of HF10-treated mice released antitumor cytokines, IL-2, IL-12, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha, after stimulation with tumor cells in vitro. The HF10-treated mice that survived their original tumor burdens rejected tumor cells upon re-challenge. These results suggested that HF10 killed HNSCC cells and induced antitumoral immunity, thereby establishing it as a promising agent for the treatment of HNSCC patients.
Project description:Immunotherapy approaches currently make their way into the clinics to improve the outcome of standard radiochemotherapy (RCT). The programed cell death receptor ligand 1 (PD-L1) is one possible target that, upon blockade, allows T cell-dependent antitumor immune responses to be executed. To date, it is unclear which RCT protocol and which fractionation scheme leads to increased PD-L1 expression and thereby renders blockade of this immune suppressive pathway reasonable. We therefore investigated the impact of radiotherapy (RT), chemotherapy (CT), and RCT on PD-L1 surface expression on tumor cells of tumor entities with differing somatic mutation prevalence. Murine melanoma (B16-F10), glioblastoma (GL261-luc2), and colorectal (CT26) tumor cells were treated with dacarbazine, temozolomide, and a combination of irinotecan, oxaliplatin, and fluorouracil, respectively. Additionally, they were irradiated with a single dose [10?Gray (Gy)] or hypo-fractionated (2?×?5?Gy), respectively, norm-fractionated (5?×?2?Gy) radiation protocols were used. PD-L1 surface and intracellular interferon (IFN)-gamma expression was measured by flow cytometry, and IL-6 release was determined by ELISA. Furthermore, tumor cell death was monitored by AnnexinV-FITC/7-AAD staining. For first in vivo analyses, the B16-F10 mouse melanoma model was chosen. In B16-F10 and GL261-luc2 cells, particularly norm-fractionated and hypo-fractionated radiation led to a significant increase of surface PD-L1, which could not be observed in CT26 cells. Furthermore, PD-L1 expression is more pronounced on vital tumor cells and goes along with increased levels of IFN-gamma in the tumor cells. In melanoma cells CT was the main trigger for IL-6 release, while in glioblastoma cells it was norm-fractionated RT. In vivo, fractionated RT only in combination with dacarbazine induced PD-L1 expression on melanoma cells. Our results suggest a tumor cell-mediated upregulation of PD-L1 expression following in particular chemoradiation that is not only dependent on the somatic mutation prevalence of the tumor entity.
Project description:Rehmannia glutinosa polysaccharide (RGP) has shown an activation of immune cells in vitro. However, the immune stimulatory effect of RGP in a mouse in vivo is not well studied. In this study, we examined the effect of RGP on dendritic cell (DC) activation and anticancer immunity in vivo. Treatments of RGP in C56BL/6 mice induced increased levels of co-stimulatory molecule expression and pro-inflammatory cytokine production in spleen DCs dependent on toll-like receptor 4 (TLR4), and those DCs promoted interferon-gamma (IFN?) production in CD4+ and CD8+ T cells. RGP also enhanced ovalbumin (OVA) antigen (Ag)-specific immune activation in tumor-bearing mice, including Ag presentation in DCs, OT-I and OT-II T-cell proliferation, migration of OT-I and OT-II T cells into the B16-OVA tumor, OVA-specific IFN? production, and the specific killing of OVA-coated splenocytes, which consequently inhibited B16-OVA tumor growth dependent on TLR4 and CD8+ T cells. Finally, the combination of RGP and self-Ag treatment efficiently inhibited CT26 carcinoma and B16 melanoma tumor growth in BLAB/c and C57BL/6 mice, respectively. These data demonstrate that RGP could be a useful adjuvant molecule for immunotherapy against cancer.
Project description:Chronic alcohol consumption has been shown to decrease the activity of natural killer (NK) cell cytolytic function and the production of various cytokines from the spleen. We have recently shown that naltrexone, an opiate receptor antagonist, when administered for a period of 2 weeks suppresses micro-opiate receptor binding but increases partial differential-opiate receptor activity in rat splenocytes. However, the effects of long-term naltrexone treatment on alcohol-induced alteration of NK cell cytolytic activity and cytokines production in splenocytes have not been determined. Male rats were pair-fed an isocaloric liquid diet or fed an ethanol-containing liquid diet for a period of 3 weeks. These rats were additionally treated after a week with a subcutaneous implant of either a naltrexone pellet or placebo pellet for 2 weeks. Splenocytes were isolated and used for determination of various cytokines interleukin (IL)-2, IL-4, and IL-6, and interferon-gamma (IFN-gamma) using enzyme-linked immunosorbent assay (ELISA), and the basal and IL-2-, IL-12-, or IL-18-induced NK cytolytic activity was measured using a standard 4-h (51)Cr release assay against YAC-1 lymphoma target cells. Ethanol consumption resulted in a reduction of the production of IL-2, IL-4, and IL-6 as well as the basal and cytokine-activated NK cell cytolytic activity and IFN-gamma production in splenocytes. Naltrexone administration increased the production of IL-2, IL-4, and IL-6 and the basal and cytokine-activated NK cell cytolytic activity and IFN-gamma production in the splenocytes of pair-fed and alcohol-fed rats. These results indicated that naltrexone treatment increases NK cell cytolytic activity and cytokine production in the spleen in vivo. Furthermore, these results identify the potential of the use of naltrexone in the treatment of immune deficiency in alcoholic and non-alcoholic patients.