Synergistic effect of interleukin-15 and interleukin-12 on antitumor activity in a murine malignant pleurisy model

 Interleukin(IL)-15, which uses IL-2 receptor (R) β and γ chains for signal transduction, shares many of the biological activities of IL-2. We examined the effects of exogenous IL-15 on protection in a murine malignant pleurisy model using BALB/c mice and syngeneic MethA fibrosarcoma (MethA). Intrapleural administration of IL-15 significantly prolonged the survival time of mice after an intrapleural inoculation of MethA, whereas the same dose of IL-2 did not. The in vivo antitumor effect of IL-15 was synergistically enhanced by additive administration of IL-12. Combination therapy of IL-15 and IL-12 protected mice from death from bloody pleural fluid. Such treatment induced marked increases in the number of CD3^-IL-2Rβ⁺ cells corresponding to natural killer (NK) cells and the production of interferon γ (IFNγ) by T cells in the thoracic exudate cells (TEC). Administration of anti-IFNγ mAb partly inhibited the protective effect of a combination of IL-15 and IL-12. A tumor-neutralizing (Winn) assay revealed that the antitumor activity of effector cells in the TEC was abrogated by treatment with anti-CD8 mAb or anti-asialoGM1 Ab plus complement. Thus, treatment with IL-15 in combination with IL-12 may enhance the activities of NK and CD8⁺ T cells in the TEC, providing strong antitumor activity against the malignant pleurisy. These results suggest that IL-15 together with IL-12 may have potential for the immunotherapy of some types of malignant pleurisy. Received: 13 July 1999 / Accepted: 3 December 1999     

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor α in mice

 There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. Received: 17 February 1997 / Accepted: 5 August 1997     

Generation of killer activity by interleukin-12 of mononuclear cells in malignant pleural effusions due to lung cancer

 In the present study, we examined the ability of interleukin (IL)-12 to generate an antitumor effect in the tumor-growing site. Mononuclear cells (MNC) were obtained from 12 malignant pleural effusions due to lung cancer in the tumor-growing site. Non-major-histocompatibility-complex-restricted killer activity, examined by 4-h ⁵¹Cr release assay against Daudi lymphoma cells as well as various lung cancer cell lines (H69 and PC-9), and in vitro production of interferon γ (IFNγ), measured by enzyme immunoassay, were investigated as mediators of antitumor effects of host cells activated by IL-12. IL-12 induced killer activity of MNC in pleural effusions (pleural MNC) dose-dependently. Moreover, pleural MNC produced a signficant amount of IFNγ in response to IL-12. The killer activities of IL-12-activated blood MNC were higher than those of pleural MNC. The supernatants of pleural effusions of these untreated patients suppressed killer induction by IL-12 of blood MNC of healthy volunteers. These observations suggest that MNC present at the site of growing tumors may act as effector cells against lung cancer in the presence of IL-12. Received: 31 December 1996 / Accepted: 10 September 1997     

MHC antigen expression by melanomas recovered from mice treated with allogeneic mouse fibroblasts genetically modified for interleukin-2 secretion and the expression of melanoma-associated antigens

Recent approaches toward the immunotherapy of neoplastic disease involve the introduction of expression-competent genes for interleukin-2 (IL-2) into autologous malignant cells. Treatment of tumor-bearing experimental animals with the IL-2-secreting cells successfully induces partial and at times complete remissions. In most instances, however, although delayed, progressive tumor growth continues. Here, certain of the characteristic of B16 melanomas (H-2^b) persisting in C57BL/6 mice (H-2^b) treated with an IL-2-secreting, melanoma-antigen-positive cellular immunogen (RLBA-IL-2 cells) are described. Unlike the melanoma cells first injected, B16 cells recovered from mice treated with RLBA-IL-2 cells were deficient in the experssion of MHC class I, but not class II determinants. Deficient MHC class I expression correlated with the cells' resistance to cytotoxic T lymphocytes (CTL) from the spleens of mice immunized with RLBA-IL-2 cells. Melanomas persisting in mice treated with non-IL-2-secreting, melanoma-antigen-positive cell constructs (RLBA-ZipNeo cells) were also deficient in the expression of MHC class I determinants, and the melanoma cells were resistant to CTL from mice immunized with RLBA-ZipNeo cells. Thus, the expression of melanoma-associated antigens rather than IL-2-secretion correlated with deficient MHC class I expression by the persistent melanomas. This point was substantiated by the expression of MHC class I antigens by melanomas persisting in mice treated with IL-2-secreting, melanoma-antigen-negative LM cells (LM-IL-2); it was equivalent to that of melanomas in untreated mice. The involvement of MHC class I antigens in the immune resistance of persistent melanoma cells from mice treated with the melanoma-autigen-positive immunogens was indicated by the effect of interferon (IFN) orN-methyl-N-nitro-N-nitrosoguanidine (MNNG) on the susceptibility of the cells to anti-melanoma CTL. Treatment of the resistant melanomas with IFN or MNNG stimulated MHC class I antigen expression and restored the cells' sensitivity to CTL from mice immunized with IL-2-secreting or nonsecreting, melanoma-antigen-positive cellular immunogens. Prior treatment of the treated cells with antibodies to MHC class I determinants inhibited the cells' susceptibility to CTL from mice immunized with RLBA-IL-2 cells.     

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells

 Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D₃ eliminated natural suppressor activity. In mice that were first treated with vitamin D₃ and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D₃/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D₃/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D₃ to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor. Received: 15 December 1995 / Accepted: 22 March 1996     

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon α and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells

 Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon γ (IFNγ), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3⁺CD56⁺ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNα but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis^y antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. Received: 9 December 1999 / Accepted: 18 May 2000     

Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1–3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20 000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 × 10⁴ cells injected i.p.) consisted of i.p. injections of 5000 or 20 000 U IL-2/mouse given on days 10–14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity. Received: 16 July 1998 / Accepted: 5 October 1998     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

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.     

Th1-cytokine induction and anti-tumor effect of 55 kDa protein isolated from Aeginetia indica L., a parasitic plant

 We have isolated a 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by affinity chromatography on an N-hydroxysuccinimide-activated Sepharose High Performance column bound with F3, a monoclonal antibody that neutralizes the cytokine-inducing and anti-tumor effect of AIL. In the present study, we examined this protein (AILb-A) for cytokine induction and anti-tumor effects by animal study, using syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. AILb-A administration resulted in markedly increased levels of Th1 cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-12 and IL-18] in the sera derived from Meth-A-bearing mice. The in vitro re-stimulation with AILb-A of splenocytes derived from AILb-A-primed mice also selectively induced Th1-type cytokines and antigen-specific killer cell activity. The neutralizing test using cytokine-specific antibodies revealed that AILb-A-induced IL-18 plays a most significant role for IFN-γ- and killer cell-inducing activities. Furthermore, IL-12 and IL-18 induced by AILb-A inhibited specifically IL-10 and IL-4 production, respectively. Finally, we examined the anti-tumor effect of AILb-A in both Meth-A-bearing BALB/c mice and Meth-A-bearing nude mice with BALB/c background. AILb-A exhibited a striking anti-tumor effect in normal BALB/c mice inoculated with Meth-A cells. In athymic nude mice, the anti-tumor effect of AILb-A was relatively weak. These findings strongly suggested that AILb-A is a potent Th1 inducer and may be a useful immunotherapeutic agent for patients with malignant diseases. Received: 27 July 2000 / Accepted: 13 March 2001     

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     

Antitumor effect of heat-killed Lactobacillus plantarum L-137 through restoration of impaired interleukin-12 production in tumor-bearing mice

 We have previously reported that heat-killed Lactobacillus plantarum L-137 is a potent inducer of interleukin-12 (IL-12) in vivo as well as in vitro in mice. In order to develop effective usage of L. plantarum L-137 for tumor immunotherapy, we examined its antitumor effect against DBA/2 mice inoculated with syngenic P388D1 tumor cells in different treatment schedules. Daily injection of L. plantarum L-137 from the day of tumor inoculation induced a steep increase in plasma IL-12 only after the first injection but not after subsequent injections, and had no effect on tumor growth and survival time. In contrast, daily injection of L. plantarum L-137 from the 7th day after tumor inoculation exerted a marked antitumor effect but such an effect was not evident in mice treated with L. plantarum L-137 twice a week from the 7th day. IL-12 production was considerably impaired at the first injection but steeply increased after the third injection in the mice injected daily with L. plantarum L-137 from the 7th day. Our results suggest that daily administration of L. plantarum L-137 is required to exert an antitumor effect at the late stages of tumor development when IL-12 production is considerably impaired. Received: 15 July 1999 / Accepted: 28 January 2000     

Postoperative administration of interleukin-12 significantly enhances the anti-tumor immune response of MBT-2 bladder cancer bearing mice

This study, using the MBT-2 murine bladder tumor model, mainly investigated the role of interleukin-12 (IL-12) in the specific antitumor immune response of a tumor-bearing host when systemically administrated after surgery. Day 17 tumor-bearing mice (D17TBM) along with non-tumor bearing naive mice were treated with daily intraperitoneal (i.p.) injection of IL-12 (0.25 microg/mouse) from day 18 to day 24 for a total of 7 doses. Their splenocytes were obtained on Day 31 for natural killer cells (NK), lymphokine activated killer cells (LAK) and cytotoxic T lymphocyte (CTL) activity assay and lymphocyte subsets phenotypic analysis. The tumor suppression effect of systemic IL-12 administration was evaluated based on the tumor outgrowth of the higher number of tumor cells rechallenged 24 hours after resectioning of the primary tumor. After evaluation on Day 31, the result of in vitro cytotoxicity assay revealed that systemic administration of IL-12 mainly enhanced the splenic LAK and CTL activities in non-tumor-primed naive mice, and the NK activity in tumor-primed D17TBM, respectively. However, in vivo administration of IL-12 with or without IL-2 failed to upgrade the proportions of either CD4+ CD44+ or CD8+ CD44+ T cells subsets in the spleens and regional inguinal lymph nodes (LNs) of both the D17TBM and naive mice. However, the splenic CD8+ CD44+ T-cell subset in the IL-12-treated D17TBM increased prominently after further culturing in the presence of IL-2 400 units/ml plus IL-12 25 ng/ml for 4 days. The fact that systemic administration of IL-12 significantly suppressed the outgrowth of Day-18 challenged tumor cells, especially in D17TBM, clearly indicates that the established specific antitumor immunity in tumor-primed D17TBM was efficiently augmented. From the results of this study, we conclude that, after surgical resection of a primary tumor, systemic administration of IL-12 can be an effective adjuvant therapy because it demonstrates a significant augmentation effect on the tumor-specific immune response in the tumor-primed host.     

Oral administration of poly-gamma-glutamate induces TLR4- and dendritic cell-dependent antitumor effect

Previously, we reported that the oral administration of high molecular mass poly-γ-glutamate (γ-PGA) induced antitumor immunity but the mechanism underlying this antitumor activity was not understood. In the present study, we found that application of high molecular mass γ-PGA induced secretion of tumor necrosis factor (TNF)-α from the bone-marrow-derived macrophages of wild type (C57BL/6 and C3H/HeN) and Toll-like receptor 2 knockout (TLR2^−/−) mice, but not those of myeloid differentiation factor 88 knockout (MyD88^−/−) and TLR4-defective mice (C3H/HeJ). Production of interferon (IFN)-γ-inducible protein 10 (IP-10) in response to treatment with γ-PGA was almost abolished in C3H/HeJ mice. In contrast to LPS, γ-PGA induced productions of TNF-α and IP-10 could not be blocked by polymyxin B. Furthermore, γ-PGA-induced interleukin-12 production was also impaired in immature dendritic cells (iDCs) from MyD88^−/− and C3H/HeJ mice. Downregulation of MyD88 and TLR4 expression using small interfering RNA (siRNA) significantly inhibited γ-PGA-induced TNF-α secretion from the RAW264.7 cells. γ-PGA-mediated intracellular signaling was markedly inhibited in C3H/HeJ cells. The antitumor effect of γ-PGA was completely abrogated in C3H/HeJ mice compared with control mice (C3H/HeN) but significant antitumor effect was generated by the intratumoral administration of C3H/HeN mice-derived iDCs followed by 2,000 kDa γ-PGA in C3H/HeJ. These findings strongly suggest that the antitumor activity of γ-PGA is mediated by TLR4. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Priming of the antitumor response promotes efficacy of interleukin-2 therapy

 We have studied the effect of active specific immunization (ASI) on the antitumor response induced by locoregional, low-dose interleukin-2 (IL-2) therapy. On day 0, mice were injected i.p. with viable, syngeneic tumor cells and with irradiated tumor cells (ASI). Low-dose IL-2 treatment was given i.p. for 5 consecutive days. ASI led to extended survival in two out of seven models tested. In these two models, enhanced efficacy was observed when both ASI and IL-2 were administered. In the five models in which ASI had no therapeutic value, ASI+IL-2 treatment was no more effective than IL-2 therapy. In the SL2 lymphoma model, use of ASI prior to IL-2 therapy given as early as days 1–5 led to at least 60% cure, whereas IL-2 therapy without ASI was only effective when administered after day 9. In the P815 mastocytoma model, however, ASI, IL-2, and the combination caused negative (suppressive) effects when administered on days 6–10. IL-2 administered on days 6–10 was therapeutically effective in this model when mice were treated with cyclophosphamide on day 6. In both the SL2 and the P815 tumor models, cured mice were specifically immune. The positive and negative effects observed were not due to the increased number of cells injected (non-specific inflammation) nor to possible antigenic alteration of the ASI cells by irradiation, as ASI with fragmented tumor cells was also effective in inducing synergy. Investigations into the underlying mechanism indicated that CD4⁺ cells play an important role. In total, the results indicate that ASI may be a good supplement to locoregional IL-2 treatment if care is taken to alleviate immunosuppressive activities. Received: 6 February 1997 / Accepted: 6 March 1997     

Characterization of B16 melanoma-specific cytotoxic T lymphocytes

A B16 melanoma-specific CD8⁺ T cell line (AB1) was established from the spleen cells of C57BL/6 mice cured of B16 melanoma with interleukin (IL)-12 treatment. The AB1 line exclusively used T cell receptor V_β11. The AB1 cells exhibited a cytolytic activity against both syngeneic B16 melanoma and allogeneic P815 mastocytoma, whereas a cold inhibition assay revealed specificity of the AB1 cells against B16 melanoma. Their lostability to kill a class I loss variant of B16 melanoma was restored by the transfection of H-2K^b gene. In addition, their interferon (IFN)-γ production was significantly suppressed by the addition of anti-H-2K^b monoclonal antibody, and RT-PCR analysis showed that the AB1 line expressed the mRNA encoding IFN-γ, but not IL-4 or IL-10. The experiment using synthetic peptides of tyrosinase-related protein-2 (TRP-2) revealed that the AB1 cells could recognize TRP-2_181–188 peptide. Moreover, the AB1 cells showed an in vivo antitumor effect against established pulmonary metastases of B16 melanoma. Overall, these results indicate that the Tc1-type V_β11 ⁺ AB1 cells exert an antitumor activity against syngeneic B16 melanoma through recognition of TRP-2_181–188 peptide in an H-2K^b-restricted manner. Received: 4 June 1998 / Accepted: 21 July 1998     

Reciprocal effects of Th1 and Treg cell inducing pathogen-associated immunomodulatory molecules on anti-tumor immunity

We have addressed the hypothesis that pathogen-associated immunomodulatory molecules may influence anti-tumor immunity through their pro- and anti-inflammatory activities and abilities to induce effector and regulatory T (Treg) cells. We found that CpG oligonucleotides (CpG) and cholera toxin (CT), which promote Th1 or Th2/Treg cell biased responses, respectively, had differential effects on tumor growth. Therapeutic peritumoral administration of CpG significantly reduced subcutaneous tumor growth and prolonged survival, whereas CT enhanced tumor growth and reduced survival. Peritumoral administration of CpG enhanced the frequency of IFN-γ-secreting and reduced IL-10-secreting CD4⁺ and CD8⁺ T cells, in the tumor and in the draining lymph nodes, whereas, CT significantly enhanced the frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells, but reduced IFN-γ-secreting T cells infiltrating the tumor. In contrast to the beneficial effect of CpG in mice with subcutaneous tumors, CpG or CT had no protective effect against tumor growth in the lungs when given therapeutically by the nasal route. However, prophylactic intranasal administration of CpG significantly reduced the number of lung metastases and this was associated with an enhanced frequency of IFN-γ-secreting CD8⁺ T cells in the draining lymph node and enhanced tumor-specific CTL responses. Our findings demonstrate that pathogen-associated molecules can either inhibit or enhance anti-tumor immunity by selectively promoting the induction of effector or regulatory T cells, and that the environment of the growing tumor influences the protective effect. Joanne Lysaght and Andrew G. Jarnicki contributed equally.     

Enhancement of anti-tumor immunity specific to murine glioma by vaccination with tumor cell lysate-pulsed dendritic cells engineered to produce interleukin-12

Aim: The aim of this study was to develop an immunotherapy specific to a malignant glioma by examining the efficacy of glioma tumor-specific cytotoxic T lymphocytes (CTL) as well as the anti-tumor immunity by vaccination with dendritic cells (DC) engineered to express murine IL-12 using adenovirus-mediated gene transfer and pulsed with a GL26 glioma cell lysate (AdVIL-12/DC+GL26) was investigated. Experimentl: For measuring CTL activity, splenocytes were harvested from the mice immunized with AdVIL-12/DC+GL26 and restimulated with syngeneic GL26 for 7 days. The frequencies of antigen-specific cytokine-secreting T cell were determined with mIFN-γ ELISPOT. The cytotoxicity of CTL was assessed in a standard 51Cr-release assay. For the protective study in the subcutaneous tumor model, the mice were vaccinated subcutaneously (s.c) with 1×10⁶ AdVIL-12/DC+GL26 in the right flanks on day −21, −14 and −7. On day 7, the mice were challenged with 1×10⁶ GL26 tumor cells in the shaved left flank. For a protective study in the intracranial tumor model, the mice were vaccinated with 1×10⁶ AdVIL-12/DC+GL26 s.c in the right flanks on days −21, −14 and −7. Fresh 1×10⁴ GL26 cells were inoculated into the brain on day 0. To prove a therapeutic benefit in established tumors, subcutaneous or intracranial GL26 tumor-bearing mice were vaccinated s.c with 1×10⁶ AdVIL-12/DC+GL26 on day 5, 12 and 19 after tumor cell inoculation. Results: Splenocytes from the mice vaccinated with the AdVIL-12/DC+GL26 showed enhanced induction of tumor-specific CTL and increased numbers of IFN-γ: secreting T cells by ELISPOT. Moreover, vaccination of AdVIL-12/DC+GL26 enhanced the induction of anti-tumor immunity in both the subcutaneous and intracranial tumor models. Conclusions: These preclinical model results suggest that DC engineered to express IL-12 and pulsed with a tumor lysate could be used in a possible immunotherapeutic strategy for malignant glioma.Korea Research Foundation Grant (KRF-2004-005-E00001).     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996