Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.     

益气补肾方药对化疗荷瘤小鼠免疫功能的影响

目的　探讨益气补肾方药 (由金匮肾气方加味人参、黄芪组成 ,以下均称方药 ,TS)对环磷酰胺 (CY)处理的荷H2 2 瘤小鼠非特异免疫功能的影响。方法　用CY处理荷H2 2 瘤小鼠 ,建立免疫力低下动物模型。用乳酸脱氢酶 (LDH)释放法分别检测NK细胞、巨噬细胞 (M)的活性 ,用RT PCR法检测白细胞介素 12 (IL 12 )mRNA在脾脏细胞中的表达。结果　TS CY组小鼠吸光度A值为 1 332± 0 5 10 ,明显高于CY组小鼠 (P <0 0 1)。TS CY组小鼠A值为 1 12 9± 0 2 80 ,明显高于CY组小鼠 (P <0 0 1)。此方药能明显提高CY所致免疫力低下小鼠NK细胞、M的活性 ,增加IL 12在脾细胞中的表达。结论　该方药可以明显提高环磷酰胺所致免疫力低下荷瘤小鼠的非特异免疫功能。     

In vitro biological activities of transmembrane superantigen staphylococcal enterotoxin A fusion protein

The bacterial superantigen staphylococcal enterotoxin A (SEA) stimulates T cells bearing certain TCR V domains when binding to MHC II molecules, and is a potent inducer of CTL activity and cytokine production. Antibody-targeted SEA such as C215 Fab-SEA and C242 Fab-SEA has been investigated for cancer therapy in recent years. We have previously reported significant tumor inhibition and prolonged survival time in tumor-bearing mice treated with a combination of both C215Fab-SEA and Ad IL-18 (Wang et al., Gene Therapy 8:542–550, 2001). In order to develop SEA as an universal biological preparation in cancer therapy, we first cloned a SEA gene from S. aureus (ATCC 13565) and a transmembrane (TM) sequence from a c-erb-b2 gene derived from human ovarian cancer cell line HO-8910, then generated a TM-SEA fusion gene by using the splice overlap extension method, and constructed the recombinant expression vector pET-28a-TM-SEA. Fusion protein TM-SEA was expressed in E. coli BL21(DE3)pLysS and purified by using the histidine tag in this vector. Purified TM-SEA spontaneously associated with cell membranes as detected by flow cytometry. TM-SEA stimulated the proliferation of both human PBLs and splenocytes derived from C57BL/6 (H-2b) mice in vitro. This study thus demonstrated a novel strategy for anchoring superantigen SEA onto the surfaces of tumor cells without any genetic manipulation.Abbreviations SEA staphylococcal enterotoxin A - TM transmembrane - NK cell natural killer cell - CTL cytotoxic T lymphocyte Drs W. Ma and H. Yu are joint corresponding authors for this article.     

Reduced tumorigenesis of EG7 after interleukin-10 gene transfer and enhanced efficacy in combination with intratumorally injection of adenovirus-mediated lymphotactin and the underlying mechanism

Although interleukin-10 (IL-10) is commonly regarded as an immunosuppressive cytokine, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating antitumor properties. Previous studies demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. In this study, we explored the tumorigenesis of EG7 cells transduced with IL-10 gene. In vivo, IL-10 gene transfer reduced tumorigenic capacity of EG7 cells and prolonged survival of the EG7 tumor-bearing mice. It was found that the cytotoxicities of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) were enhanced. Assessment of the immune status of the animals showed prevalence of a systemic and tumor-specific Th2 response (high levels of IL-4 and IL-10). To improve the therapeutic efficacy, we combined with intratumoral injection of adenovirus-mediated lymphotactin (Ad-Lptn) into the overestablished EG7 tumor model. More significant inhibition of tumor growth were observed in EG7 tumor-bearing mice that received combined treatment with IL-10 and Lptn gene than those of mice treated with IL-10 or Lptn gene alone. The highest NK cells and CTL activity was induced in the combined therapy group, increasing the production of IL-2 and interferon-γ (IFN-γ) significantly but decreasing the expression of immune suppressive cells (CD4⁺Foxp3⁺ Treg cells and Gr1⁺CD11b⁺ MDSCs). The necrosis of tumor cells was markedly observed in the tumor tissues, accompanying with strongest expression of Mig (monokine induced by interferon-gamma) and IP-10 (interferon-inducible protein 10), weakest expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2). In vivo, depletion analysis demonstrated that CD8⁺ T cells and NK cells were the predominant effector cell subset responsible for the antitumor effect of IL-10 or Lptn gene. These findings may provide a potential strategy to improve the antitumor efficacy of IL-10 and Lptn.     

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8⁺ T or CD4⁺ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18. Received: 7 May 1998 / Accepted: 27 May 1999     

IL-2 plasmid therapy of murine ovarian carcinoma inhibits the growth of tumor ascites and alters its cytokine profile

We have evaluated whether i.p. murine ovarian tumors could be treated with an IL-2 plasmid DNA complexed with the cationic lipid, (+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2, 3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (DMRIE/DOPE). Reporter gene studies were initially conducted in which mice bearing i.p. murine ovarian teratocarcinoma (MOT) were injected i.p. with reporter gene plasmid DNA (pDNA):DMRIE/DOPE. Histochemical analyses revealed that transfection occurred primarily in the tumor cells of the ascites, with only a minority of other ascitic cells or surrounding tissues transfected. IL-2 levels in the MOT ascites were determined after i. p. injection of either IL-2 pDNA:DMRIE/DOPE or recombinant IL-2 protein. IL-2 was detected in tumor ascites for up to 10 days after a single i.p. injection of IL-2 pDNA:DMRIE/DOPE, but was undetectable 24 h after a single i.p. injection of IL-2 protein. In an antitumor efficacy study, MOT tumor-bearing mice injected i.p. with IL-2 pDNA:DMRIE/DOPE on days 5, 8, and 11 after tumor cell implant had a significant inhibition of tumor ascites (p = 0.001) as well as a significant increase in survival (p = 0.008). A cytokine profile of the MOT tumor ascites revealed that mice treated with IL-2 pDNA:DMRIE/DOPE had an IL-2-specific increase in the levels of IFN-gamma and GM-CSF. Taken together, these findings indicate that i. p. treatment of ovarian tumors with IL-2 pDNA:DMRIE/DOPE can lead to an increase in local IL-2 levels, a change in the cytokine profile of the tumor ascites, and a significant antitumor effect.     

Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

Virulizin, a novel biological response modifier, has demonstrated significant antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. The significant role of macrophages and NK (Natural killer) cells was implicated in the antitumor mechanism of Virulizin where expansion as well as increased activity of macrophages and NK cells were observed in mice treated with Virulizin. Depletion of macrophages compromised Virulizin-induced NK1.1⁺ cell infiltration into xenografted tumors and was accompanied by reduced antitumor efficacy. In the present study, involvement of macrophages in NK cell activation was investigated further. We found that depletion of NK cells in CD-1 nude mice by anti-ASGM1 antibody significantly compromised the antitumor activity of Virulizin. Cytotoxicity of NK cells isolated from Virulizin-treated mice was enhanced against NK-sensitive YAC-1 cells and C8161 human melanoma cells, but not against NK-insensitive P815 cells. An increased level of IL-12 was observed in the serum of mice treated with Virulizin. IL-12 mRNA and protein levels were also increased in peritoneal macrophages isolated from Virulizin-treated mice. Moreover, Virulizin-induced cytotoxic activity of NK cells isolated from the spleen was abolished when an IL-12 neutralizing antibody was co-administered. In addition, depletion of macrophages in mice significantly impaired Virulizin-induced NK cell cytotoxicty. Taken together, the results suggest that Virulizin induces macrophage IL-12 production, which in turn stimulates NK cell-mediated antitumor activity.     

Immunotherapy of intraperitoneal cancer with interleukin 2 and lymphokine-activated killer cells reduces tumor load and prolongs survival in murine models

The control of malignancy disseminated within the peritoneal cavity is an important problem in the management of low-grade gastrointestinal and ovarian neoplasms. A model of peritoneal carcinomatosis in the mouse was used to investigate the potential of lymphokine-activated killer (LAK) cells and exogenous interleukin 2 (IL-2) to control intraperitoneal tumor. LAK cells are splenocytes activated in vitro by IL-2. C57BL/6 mice were injected intraperitoneally with a lethal inoculum of syngeneic MCA-105 tumor. Three days later, the established tumor was treated with adoptively transferred LAK cells and/or exogenous IL-2 administration. LAK cells alone were ineffective in reducing intraperitoneal tumor. Administration of IL-2 alone resulted in limited tumor reduction. Treatment with exogenous IL-2 in conjunction with LAK cells resulted in the greatest reduction of intraperitoneal tumor. The larger the number of LAK cells given, the greater the reduction in tumor. Frequent intraperitoneal bolus administration of IL-2 was more effective than a single daily intraperitoneal injection and intraperitoneal administration of IL-2 and LAK was more effective than systemic treatments. Marked prolongation of life was seen in mice treated with LAK cells plus exogenous IL-2. We conclude that intraperitoneal LAK cells plus exogenous IL-2 is an effective treatment regimen for reducing intraperitoneal tumor in this murine model.     

Effects of granulocyte-colony-stimulating factor and interleukin-2 on ascites formation and the survival time of nude mice bearing human ovarian cancer cells

 The aim of this study was to elucidate the effect of intraperitoneal (i.p.) instillations of granulocyte-colony-stimulating factor (G-CSF) and/or interleukin-2 (IL-2) on ascites formation and the survival time of nude mice with malignant ascites, produced by i.p. inoculation of human ovarian cancer cells. When the nude mice were treated with medium alone, ascites was observed in all mice 28 days after tumor inoculation. When the mice were treated with cis-diamminedichloroplatinum(II) (cisplatin) alone, G-CSF alone or IL-2 alone, it took 35 days for the ascites to form in all mice. When cisplatin was combined with G-CSF or IL-2, one of ten mice did not form ascites during the observation period. Surprisingly, when G-CSF and IL-2 were simultaneously administered, ascites formation was not observed in any mice. Although i.p. treatment with cisplatin alone significantly prolonged the survival time, compared to medium alone, the lytic activity of spleen cells against HRA cells was significantly suppressed. When G-CSF or IL-2 was combined with cisplatin, the suppression by cisplatin was eliminated and subsequently resulted in a prolongation of the survival time. When G-CSF was combined with IL-2, both the peritoneal and splenic macrophages/monocytes were stimulated and the splenic lytic activity was about double that following treatment with G-CSF alone on IL-2 alone, suggesting that complete inhibition of ascites formation results not only from a significant increase of the peritoneal macrophages but also from enhancement of the lytic activity. Two mice, died from dissemination of tumor in the abdominal cavity, but eight mice survived without tumor for more than 90 days. As confirmed by monitoring body weight and hematocrit, G-CSF and IL-2 seemed to have no adverse effect. From these results, we conclude that a combination therapy with G-CSF and IL-2 might be of clinical use for inhibiting large amounts of ascites, which may inhibit therapeutic effects for ovarian cancer patients. Received: 20 May 1996 / Accepted: 19 September 1996     

Oncolytic Adenovirus Expressing IL-23 and p35 Elicits IFN-γ- and TNF-α-Co-Producing T Cell-Mediated Antitumor Immunity

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4⁺ and CD8⁺ T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

Induction of an effective anti-tumor immune response and tumor regression by combined administration of IL-18 and Apoptin

Immunization strategies using plasmid DNA can potentially improve humoral and cellular immune responses that protect against cancer and infectious diseases. The chicken anemia virus-derived Apoptin protein exhibits remarkable specificity in its ability to induce apoptosis in tumor cells, but not in normal diploid cells. Interleukin-18 (IL-18) is a Th1-type cytokine that has demonstrated potential as a biological adjuvant in murine tumor models. In this study, we analyzed the anti-tumor potential and mechanism of action of simultaneous Apoptin and IL-18 gene transfer in C57BL/6 mice bearing Lewis lung carcinoma (LLC). Here we report that the growth of established tumors in mice immunized with pAPOPTIN in conjunction with pIL-18 was significantly inhibited compared with the growth of tumors in mice immunized with the empty vector (EV) or pAPOPTIN alone. Furthermore, the immunization of mice with pAPOPTIN in conjunction with pIL-18 elicited strong natural killer activity and LLC tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro. In addition, T cells from lymph nodes of mice vaccinated with pIL-18 or pAPOPTIN + pIL-18 secreted high levels of the Th1 cytokine IL-2 and IFN-γ, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with Apoptin together with IL-18 may be a novel and powerful strategy for cancer immunotherapy.     

Generation propagation, and targeting of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus recombinant IL-2. An efficient strategy for adoptive tumor immunotherapy.

We developed a culture system for the rapid generation of CD4+ T cells that have both helper and killer functions. CD4+ T cells isolated from human PBL did not proliferate or develop significant cytotoxicity when treated with rIL-2 because of the lack of p75 IL-2R expression. However, culture of isolated CD4+ T cells with immobilized anti-CD3 mAb plus rIL-2 resulted in a marked proliferation (500-fold increase in 14 days) of CD4+ T cells. The proliferating CD4+ T cells produced IL-2 (92 U/ml) and showed strong cytotoxicity against OKT3 hybridoma cells and Daudi, K562, and U937 tumor cells in an anti-CD3 mAb-dependent manner. The CD4+ T cells contained significant amounts of cytolytic granule-related proteins such as serine esterase and perforin. Activated CD4+ helper/killer cells can be generated from both healthy donors and tumor patients and can be propagated in vitro for 14 to 35 days by biweekly restimulation with immobilized anti-CD3 mAb plus rIL-2. This culture yielded about 20,000-fold increase in cell number after a 21-day culture. Bispecific antibody containing anti-CD3 and anti-glioma Fab components enhanced the cytotoxicity of activated CD4+ helper/killer cells against IMR32 glioma cells. Moreover, the activated CD4+ helper/killer cells showed both helper and antitumor activity in vivo and prevented growth of anti-CD3 hybridoma cells in nude mice whether or not IL-2 was administered. These results indicate that anti-CD3 mAb plus IL-2-activated CD4+ helper/killer cells may provide an effective strategy for adoptive tumor immunotherapy of cancer.     

High density of IL-17-producing cells is associated with improved prognosis for advanced epithelial ovarian cancer

Interleukin (IL)-17 is the signature cytokine of T helper 17 cells. The role of IL-17 in the tumor microenvironment is still controversial. Few studies describing IL-17 expression in ovarian cancer have been reported. We have therefore analyzed the in situ tumor expression of IL-17 in advanced ovarian cancer and the possible correlation of IL-17 expression with tumor-associated macrophages (TAMs) and with survival in advanced ovarian cancer. Clinical data of 104 patients with stage III–IV epithelial ovarian cancer at the Sun Yat-sen University Cancer Center between 2000 and 2008 were retrospectively reviewed. Immunohistochemical staining of IL-17 and CD163 (marker for TAMs) was performed. Our data showed that levels of IL-17 were significantly increased in ovarian cancer compared with normal ovarian tissues (P<0.001). The high IL-17 expression group included more patients with grade 1 tumors than the low IL-17 expression group (P=0.042). High IL-17 expression correlated with improved progression-free survival (PFS) in advanced ovarian cancer (P<0.001). However, no significant difference was observed in overall survival between the high and low IL-17 expression groups. Multivariate analysis revealed that the density of IL-17-producing cells was a positive prognostic factor for PFS (P=0.001). Moreover, a positive correlation between the density of IL-17-producing cells and TAMs was identified (r=0.354, P<0.001). Our results indicate that the infiltration of IL-17-producing cells might contribute to improved PFS in advanced ovarian cancer. Our findings provide a new insight into the complex role of IL-17 in the tumor microenvironment of ovarian cancer.     

Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection

Interleukin (IL)-10 is an anti-inflammatory cytokine known to modulate the outcome of sepsis by decreasing pro-inflammatory cytokine production, including IL-12, a main activator of natural killer (NK) cells. We hypothesized that neutralization of IL-10 would increase NK and natural killer T (NKT) cell activation through increased IL-12 in a mouse model of bacterial peritonitis. NK and NKT cell activations were measured by CD69 expression on NK1.1+/CD3- and NK1.1+/CD3+ cells after cecal ligation and puncture (CLP). NK cells were significantly more activated in mice treated with anti-IL-10 antibodies, whereas no such effect was observed in NKT cells. Similarly, intracellular interferon gamma (IFN-gamma) levels were increased in NK cells of anti-IL-10-treated mice, but not in NKT cells. IL-12 and IL-18 levels were increased in both CLP groups, but in anti-IL-10-treated mice, early IL-12 and late IL-18 levels were significantly higher than in controls. Survival at 18 h after CLP was lower in anti-IL-10 mice, which was associated with increased liver neutrophil accumulation. In summary, these data show an activating effect of IL-10 on NK, but not on NKT cells after CLP, which corresponded with decreased survival, higher IFN-gamma production, and increased remote organ neutrophil accumulation. These effects were not mediated by IL-12 and IL-18 alone, and reinforce a role for NK cells in remote organ dysfunction following peritonitis.     

Interferon-α and interleukin-12 gene therapy of cancer: interferon-α induces tumor-specific immune responses while interleukin-12 stimulates non-specific killing

Cytokine gene therapy is applied in clinical studies of tumors, and IFN-alpha and IL-12 are widely used for cancer immunotherapy. Using a poorly immunogenic murine colorectal cancer cell line, MC38, we compared antitumor effects of IFN-alpha and IL-12. Transduced MC38 cell lines expressing IFN-alpha or IL-12 (MC38-IFNalpha or MC38-IL12, respectively) were established using retroviral vectors. Transduction of IFN-alpha or IL-12 gene to MC38 cells significantly reduced tumorigenicity in immunocompetent mice. When tumor-free mice initially injected with MC38-IFNalpha or MC38-IL12 cells were reinjected contralaterally with wild-type MC38 cells (MC38-WT) after 35 days, 7 of 12 or 2 of 12 mice rejected MC38-WT cells, respectively. In therapy-model mice with established tumor derived from MC38-WT cells, inoculation of gene-transduced cells significantly suppressed growth of the tumor in MC38-IFNalpha-inoculated groups, but not in the IL-12-inoculated group. Immunohistologic and flow cytometric analyses showed marked infiltration of CD8(+) cells in wild-type tumors of mice inoculated with IFN-alpha-expressing cells. Leukocyte-depletion experiments implicated CD8(+) T cells in tumor rejection induced by IFN-alpha-transduction; both CD8(+) T cells and natural killer cells were implicated in the more modest antitumor effect from IL-12 expression. To investigate induction of tumor-specific immune responses, we stimulated splenocytes from tumor-free mice twice in vitro with genetically modified MC38 cells. In vitro stimulations with MC38-IFNalpha cells induced definite MC38-specific lysis, but not stimulations with MC38-IL-12 cells. Injecting combination of MC38-IFNalpha and MC38-IL-12 cells caused an additive antitumor effect in the therapy model. These data suggested that IFN-alpha induces cytotoxic T lymphocytes and elicits long-lasting tumor-specific immunity, whereas IL-12 seems to stimulate non-specific killing. With additional refinements, combined IFN-alpha and IL-12 gene therapy might warrant clinical trials.     

Interleukin-21 expanded NKDC in vitro reduces the B16F10 tumor growth in vivo

Innate immunity to tumors is mediated mainly by natural killer cells (NKs) and dendritic cells (DCs). The function of these cells is coordinated by cytokines produced during the inflammatory process. NK cells are highly active against tumors, being an important source of IFN-γ. Natural killer dendritic cells (NKDCs) were recently identified as a group of hybrid cells; some studies claim that they have lytic activity, produce IFN-γ and can also stimulate antigen-specific T cells. Interleukin 21 (IL-21) regulates the proliferation capacity and cytotoxicity of NK and T cells. The main objective of this study was to investigate if IL-21 influences the frequency of NKDCs in vitro as well as IFN-γ production and also to verify if these cells could enhance the antitumor activity against B16F10 tumor model in vivo. Splenocytes from C57BL/6 mice were isolated and the DC were enriched by immunomagnetic beads and cultured for four days with recombinant IL-21 (10, 20, 40 or 100 ng/ml). NKDC population was characterized as CD11c^low/medB220⁺NK1.1⁺. Expanded cells were used to treat B16F10 tumor bearing mice and tumor growth was compared between the doses of IL-21 10 ng/ml and 20 ng/ml. The results indicate that IL-21 increases the expansion of splenic NKDCs in vitro in doses of 10 ng/ml and 20 ng/ml and these cells produce IFN-γ. In vivo, cells expanded with IL-21 and injected directly into the growing tumor efficiently reduced the tumor size. Together, these results showed for the first time that IL-21 influences the biology and the effector activity of NKDCs.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

IL-8 reduced tumorigenicity of human ovarian cancer in vivo due to neutrophil infiltration

Paclitaxel is a frontline therapy for ovarian cancer. Our laboratory has shown that paclitaxel induces IL-8, a member of the C-X-C family of chemokines, in subsets of human ovarian cancer cells. However, the critical issue concerns the biological significance of this chemokine in human ovarian cancer. To study the influence of IL-8 on tumor growth, human ovarian cancer cell lines were transfected with an expression vector for human IL-8 and tested for their ability to form tumors in nude mice. IL-8 expression by the transfected cells did not alter their growth properties in vitro. In contrast, tumor growth in vivo was significantly attenuated in animals receiving IL-8-expressing cells when compared with mice injected with control cells. As additional evidence that IL-8 is a crucial factor in tumor growth, it was noted that ovarian cell lines in which constitutive IL-8 expression is elevated did not form tumors. Injection of neutralizing Ab to IL-8 reverted the phenotype and caused tumor growth in vivo. Examination of tissue from the inoculation site revealed a dramatically elevated cellularity, containing neutrophils and macrophages, in mice receiving IL-8-expressing tumor cells. These results suggest that IL-8 production by human ovarian tumor cells can play a role in reducing the rate of tumor growth; this effect may be mediated by the increased targeting of neutrophil and other mononuclear cells to the tumor injection site. These studies indicate a role for IL-8 in ovarian cancer control and suggest that chemotherapy-induced IL-8 may have a positive role in controlling tumor growth.     

Gene Therapy against Murine Melanoma B16F10-Nex2 Using IL-13Ralpha2-Fc Chimera and Interleukin 12 in Association with a Cyclopalladated Drug

Interleukin 13 (IL-13) is immunoregulatory in many diseases, including cancer. The protective or suppressive role of CD1-restricted natural killer T cells (NKT cells) in tumor immunosurveillance and immunity is well documented. Interleukin 12 (IL-12) can activate type I NKT cells to produce interferon-gamma (IFN-gamma), whereas type II NKT cells may produce IL-13. The high-affinity chain of IL-13Ralpha2 may act as negative inhibitor, suppressing the action of IL-13 and helping to maintain tumor immunosurveillance. We constructed an mIL-13Ralpha2-Fc chimera in a eukaryotic expression vector and confirmed the identity of the recombinant protein by immunoblot analysis and binding to IL-13 in chemiluminescent ELISA. Such DNA vaccine was tested against syngeneic B16F10-Nex2 murine melanoma. In vivo experiments showed a protective effect mediated by high production of IFN-gamma and down-regulation of anti-inflammatory interleukins mainly by NKT 1.1(+) T cells. Biochemoterapy in vivo with plasmid encoding mIL-13Ralpha2-Fc in association with plasmid encoding IL-12 and the 7A cyclopalladated drug led to a significant reduction in the tumor evolution with 30% tumor-free mice. We conclude that IL-12 gene therapy, followed by continuous administration of IL-13Ralpha2-Fc gene along with 7A-drug has antitumor activity involving the high production of proinflammatory cytokines and low immune suppression, specifically by NK1.1(+)T cells producing IL-13 and IL-10.     

Sequential immunotherapy using interleukin-1 followed by interleukin-2 of ascitic MOPC104E-bearing mice

This study shows that intraperitoneal injection of interleukin-1 (IL-1), followed by interleukin-2 (IL-2), can effectively eradicate murine ascitic tumor cells. This antitumor effect of IL-1 and IL-2 was abolished when administration of IL-2 preceded that of IL-1. Solid tumors inoculated subcutaneously (s.c.) into the back of mice were also sensitive to this combined i.p. therapy, indicating a systemically-operating antitumor mechanism. Splenocytes from tumor-bearing mice treated with IL-1 followed by IL-2 showed a strong tumor-neutralizing activity. The population responsible proved to be Lyt2.2 (CD8)-positive cells.Abbreviations IL interleukin - LAK lymphokine activated killer - LU lytic unit - MST median survival time - SE sonicated tumor extract