Efficient inducation of local and systemic antitumor immune response by liposome-mediated intratumoral co-transfer of interleukin-2 gene and interleukin-6 gene.

Interleukin 2 (IL-2) expressing plasmid and interleukin 6 (IL-6)-expressing plasmid were encapsulated in liposome and administrated intratumoraly into tumor-bearing mice 4 days after subcutaneous inoculation of B16F10 melanoma cells. The results showed that treatment of tumor-bearing mice with IL-2 gene or IL-6 gene transfer inhibited the growth of subcutaneous tumor and prolonged the survival of tumor-bearing mice significantly when compared with the treatment of PBS or control gene transfer mediated by liposome (P < 0.01). Combined transfer of IL-2 gene and IL-6 gene was found to elicit inhibitory effects on the growth of B16F10 tumor more significantly and prolonged the survival period of tumor-bearing mice more obviously. We investigated the local immunity in tumor microenvironment and found that IL-2 and IL-6 gene transfer could significantly increase the expression of lymphocyte function-associated antigen-1 on tumor infiltrating lymphocytes (TIL) and MHC-I molecule on tumor cells freshly isolated from the tumor mass. The NK and CTL activity of TIL increased markedly after the combined transfer of these two cytokine genes. We also observed the systemic antitumor immune response in the tumor-bearing mice and demonstrated that NK and CTL activity of splenocytes and the production of IL-2, tumor necrosis factor and interferon-gamma from splenocytes increased obviously in mice after the combined transfer of IL-2 and IL-6 gene. In conclusion, local and systemic antitumor immunity of the tumor-bearing host could be induced efficiently after the combined gene transfer. The enhanced specific and non-specific antitumor immunity might be responsible for the more potent antitumor effects of the combined gene therapy.     

Phenotype of syngeneic tumor-specific cytotoxic T-lymphocytes and requirements for their in vitro generation from tumor-bearing host and immune spleens

Cells required for the in vitro generation of syngeneic cytotoxic T-lymphocytes (CTL) against the P815 mastocytoma in the DBA/2 mouse strain were investigated. For both immune and tumor-bearing host spleen cells, CTL effector cells were eliminated by treatment with anti-Thy1.2, anti-Lyt1.1, or anti-Lyt2.1 and C', but were resistant to anti-L3T4 (GK1.5). Thus, CTL effectors (and their precursors) were Lyt1+2+, L3T4-. However, P815-specific CTL could not be generated in the absence of L3T4+ cells, whose function could be replaced with exogenous interleukin-2 (IL-2). When monoclonal antibodies against L3T4 were added to mixed leukocyte tumor cultures, CTL generation was markedly inhibited. Depletion of accessory cells also led to a marked reduction in CTL generation, which could be restored to control levels by adding adherent cells from normal spleens or with exogenous IL-2, but not with IL-1. Thus, accessory cells are apparently required to present the tumor antigens of this Ia-negative tumor to T-helper cells.     

Transforming growth factor beta subverts the immune system into directly promoting tumor growth through interleukin-17

Overexpression of the immunosuppressive cytokine transforming growth factor beta (TGF-beta) is one strategy that tumors have developed to evade effective immunesurveillance. Using transplantable models of breast and colon cancer, we made the unexpected finding that CD8+ cells in tumor-bearing animals can directly promote tumorigenesis, by a mechanism that is dependent on TGF-beta. We showed that CD8+ splenocytes from tumor-bearing mice expressed elevated interleukin (IL)-17 when compared with naive mice, and that CD8+ T cells could be induced to make IL-17 on addition of TGF-beta and IL-6 in vitro. Treatment of mice with anti-TGF-beta antibodies in vivo reduced IL-17 expression both in the tumor and the locoregional lymph nodes. Although IL-17 has not previously been shown to act as a survival factor for epithelial cells, we found that IL-17 suppressed apoptosis of several tumor cell lines in vitro, suggesting that this altered T-cell polarization has the potential to promote tumorigenesis directly, rather than indirectly through inflammatory sequelae. Consistent with this hypothesis, knockdown of the IL-17 receptor in 4T1 mouse mammary cancer cells enhanced apoptosis and decreased tumor growth in vivo. Thus, in addition to suppressing immune surveillance, tumor-induced TGF-beta may actively subvert the CD8+ arm of the immune system into directly promoting tumor growth by an IL-17-dependent mechanism.     

Autologous high-killing cytotoxic T lymphocytes against human lung cancer are induced using interleukin (IL)-1beta, IL-2, IL-4, and IL-6: possible involvement of dendritic cells.

Although CTLs bear main immune responses in human tumors, stable CTL clones against human lung cancer have rarely been generated. Our previous study demonstrated efficient autologous CTL induction in human gastric cancer and glioblastoma by cytokine combination of interleukin (IL)-1beta (167 IU/ml), IL-2 (67 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml). In this study, we demonstrated successful induction of autologous stable CTLs in five of six patients with lung adenocarcinoma from mixed-lymphocyte tumor culture using this cytokine combination. All CTLs revealed potent and specific killing activity against autologous target cells (over 75% in CD8+ CTLs and over 50% in CD4+ CTLs at an E:T ratio of 10 for 24 h). Using a series of antibodies, CD8+ CTLs showed to recognize tumor-specific antigens of lung cancer cells through HLA class I. In the separate experiments, failure of CTL induction from monocyte-depleted peripheral blood mononuclear cells and appearance of cells with characteristics of dendritic cells from adherent peripheral blood mononuclear cells in the culture of the same concentration of IL-1beta, IL-4, and IL-6 indicated that CTLs can be efficiently generated by this cytokine combination via possible dendritic cell induction. This is the first study of an efficient and reproducible in vitro CTL induction against human lung cancer.     

Immunotherapy with recombinant SFV-replicons expressing the P815A tumor antigen or IL-12 induces tumor regression

Replicons based on alphaviruses are emerging as candidate vectors for vaccination and gene therapy purposes. We have reported previously that mice vaccinated with a recombinant Semliki Forest virus (a member of the alphavirus genus) carrying the gene encoding the P815A tumor antigen (rSFV/E-P1A) were protected against a lethal challenge with the P815 tumor. In this study we investigated the anti-tumor therapeutic efficacy of rSFV/E-P1A or rSFV expressing the cytokine interleukin 12 (rSFV/IL12) on Day 5-established tumors. The results show that both antigen-specific and cytokine-mediated rSFV treatments exhibited a significant effect on P815 tumor growth, by delaying tumor progression and even inducing complete tumor regressions in several mice. The therapeutic potency of these vectors was dependent on the size of the treated tumor, as treatment of mice bearing larger tumors showed lower efficacy. In addition, rSFV treatment resulted in long-term immunity as observed by the lack of tumor recurrence in the majority of tumor-regressing mice after rechallenge with the tumor. Furthermore, the anti-tumor therapeutic effect was only achieved by local intratumoral injection of rSFV, as treatment by injection in the contralateral site resulted in tumor progression comparable to control-untreated mice. Accordingly, expression of a rSFV-RNA was localized to the tumor and draining lymph node. These results further demonstrate the potential of rSFV replicons as tumor therapeutic agents.     

Activation of suppressor cells by syngeneic tumor transplants in mice.

Spleen cells from mice with syngeneic tumor transplants are hyporesponsive in mixed-lymphocyte culture. The hyporesponsiveness is due to the activation of suppressor cells. Spleen from tumor-bearing mice, treated with mitomycin and added to normal mixed lymphocyte culture (with responding cells syngeneic to the added cells), inhibits proliferation of the responding cells. Suppressor activity in the spleen cells can be detected as early as 5 days after s.c. transplantation of P-815 mastocytoma into DBA/2 mice. Tumor cells placed in cell-impermeable i.p. diffusion chambers can also activate splenic suppressor cells. Suppressor cells can be activated in syngeneic mice by (DBA/2) P-815 cells, by (C3H) L25-cells, or by recent (C57BL/6) methylcholanthrene-induced tumors. The latter tumors retain their ability to activate suppressor cells after passaging in syngeneic mice. Only one tumor, induced with methylcholanthrene in DBA/2 mice, failed to activate suppressor cells. Suppressor activity in the spleen cells from mice with 20-day s.c. tumor transplants is not reduced after removal of glass-adherent cells. Suppressor activity is significantly decreased after removal of thymus-derived cells with anti-theta treatment and complement.     

联合自杀基因与IL-2基因转移疗法的抗肿瘤效果及其抗肿瘤免疫应答的诱导作用

单用自杀基因疗法或单用细胞因子基因疗法抗肿瘤效果不理想,本研究中我们观察了大肠杆菌胞嘧啶脱胺酶(CD)基因与白细胞介素2(IL-2)基因联合转移对荷瘤小鼠的治疗效果及其对抗肿瘤免疫的诱导作用。复制荷瘤小鼠模型后在荷瘤部位注射表达CD基因的重组腺病毒(AdCD)及表达小鼠IL-2基因的重组腺病毒(AdIL2),并连续10天、每天1次腹腔注射5氟胞嘧啶(5FC)对荷瘤小鼠进行治疗。结果表明,AdCD/5FC/AdIL2联合基因治疗能显著抑制荷瘤小鼠皮下肿瘤的生长,并明显延长其生存期(P<0.01)。联合基因治疗组小鼠肿瘤细胞发生明显的坏死,瘤内及瘤周有大量的炎性细胞浸润,瘤内CD4~ 和CD8~ T细胞明显增加,脾细胞NK和CIL杀伤活性明显高于单用AdCD/5FC、对照病毒AdLacZ/5FC或PBS组。实验结果表明,联合应用自杀基因与细胞因子基因治疗可更有效诱导机体的抗肿瘤免疫反应,从而更显著地抑制荷瘤小鼠肿瘤的生长。     

Induction of a cytolytic T-cell response in mice with a recombinant adenovirus coding for tumor antigen P815A

We investigated the efficacy of a recombinant adenovirus in inducing a cytolytic T-lymphocyte (CTL) response in mice against tumor antigen P815A, which is present on mouse mastocytoma P815. The recombinant adenoviral vector (Adeno.PIA) contained the sequence coding for the antigenic nonapeptide which binds to the H-2.L^d molecule to form antigen P815A. We verified that murine cells infected in vitro with Adeno.PIA were lysed by an anti-P815A CTL clone. Mice then received a single intradermal injection of Adeno.PIA, and after a few weeks their spleen cells were stimulated in vitro with tumor cells expressing antigen P815A. An anti-P815A CTL response was observed with the spleen lymphocytes of nearly all the mice, providing the lymphocytes were re-stimulated in vitro with cells expressing both P815A and co-stimulatory molecule B7.1. When the stimulatory cells did not express B7.1, a specific CTL response was observed in only 45% of the mice, and it was less intense. The Adeno.PIA viral vector was unable to raise an anti-P815A response in mice that had been previously infected with a recombinant adenovirus carrying the β-galactosidase gene or with a defective adenovirus. We conclude that adenoviral vectors may be very useful for the priming of cytolytic T-cell responses directed against human tumor antigens. Other modes of immunization may be necessary to boost the responses induced with adenoviral vectors. © 1996 Wiley-Liss, Inc.     

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VI. Occasional escape from host rejection due to antigen-loss secondary variants

After mutagenesis of mouse mastocytoma P815, it is possible to obtain variant (tum-) clones that are almost always rejected by syngeneic DBA/2 mice. Most tum- clones express new variant-specific antigens that can be detected by cytolytic T cells (CTL). Occasionally, mice injected with tum- variants eventually develop progressive tumors. Cells from these tumors were analyzed for antigen expression with variant-specific and P815-specific CTL clones. Out of 13 tumors examined, 11 were composed of cells that had clearly lost a variant-specific antigenic determinant. These results indicate that tum- variants induce a specific host rejection response which usually results in complete elimination of the variant cells, but that occasional antigen-loss constitutes an important mechanism of escape. The loss of a variant-specific antigenic determinant from one tum- clone that had escaped rejection allowed the detection of a residual variant-specific determinant. This was demonstrated by isolating a new CTL clone that lysed both the original tum- clone and its antigen-loss variant, but not other P815 targets. Thus, some complex transplantation antigens can be separated into independent determinants by using antigen-loss variants.     

Therapeutic effects of adoptive splenocyte transfer following in situ AdIL-12 gene therapy in a mouse prostate cancer model

We developed a preclinical prostate cancer model to study the feasibility of adoptive immunotherapy for residual tumor following neo-adjuvant in situ adenoviral-vector-mediated interleukin 12 (AdIL-12) gene therapy. Splenocytes were obtained from mice with orthotopic 178-2 BMA metastatic mouse prostate cancers treated previously with AdIL-12, or a vector with the IL-12 genes plus the costimulatory gene B7-1 (AdIL-12/B7), or a control gene (Adbetagal). The splenocytes were subsequently injected intravenously into syngeneic mice bearing orthotopic 178-2 BMA tumors generated 3 days previously. Significant orthotopic tumor growth suppression was achieved with splenocytes derived from mice whose tumors had been injected with AdIL-12 compared to splenocytes from control Adbetagal mice (P = 0.0005) and splenocytes from AdIL-12/B7-treated mice significantly suppressed spontaneous lung metastases compared to splenocytes from control mice (P = 0.0356). Adoptive transfer of splenocytes from either AdIL-12 (P = 0.004) or AdIL-12/B7 (P = 0.009)-treated mice significantly prolonged survival relative to controls. Transfer of NK and tumor-specific CTL activities was detected and depletion of CD4+ and CD8+ T cells by in vitro antibody-mediated complement lysis of the splenocytes prior to injection abrogated the effects. Systemic IL-12 administration delivered by intramuscular AdIL-12 injection enhanced the antitumor effects of adoptive splenocyte transfer and boosted the CTL response. Our data provide evidence that this form of adoptive immunotherapy can enhance the effectiveness of neo-adjuvant in situ IL-12 gene therapy in cases of persistent malignancy.     

Transforming growth factor beta inhibits the antigen-presenting functions and antitumor activity of dendritic cell vaccines

Dendritic cell (DC)-based vaccines have exhibited minimal effectiveness in treating established tumors, likely because of factors present in the tumor microenvironment. One such factor is transforming growth factor beta (TGF-beta), a cytokine that is produced by numerous tumor types and has been demonstrated to impair DC functions in vitro. We have evaluated the effect of TGF-beta on the immunostimulatory activities of DCs. We demonstrate that TGF-beta exposure inhibits the ability of DCs to present antigen, stimulate tumor-sensitized T lymphocytes, and migrate to draining lymph nodes. Neutralization of TGF-beta using the TGF-beta-neutralizing monoclonal antibody 2G7 enhanced the ability of DC vaccines to inhibit the growth of established 4T1 murine mammary tumors. Treatment of 4T1 tumors transduced with the antisense TGF-beta transgene (4T1-asT) with the combination of DC and 2G7 monoclonal antibody inhibited tumor growth and resulted in complete regression of tumors in 40% of the mice. These results demonstrate that neutralization of TGF-beta in tumor-bearing mice enhances the efficacy of DC-based vaccines.     

Tumor-infiltrating dendritic cell subsets of progressive or regressive tumors induce suppressive or protective immune responses

Tumor-infiltrating dendritic cells (TID) have an ambivalent role in regulation of tumor regression or growth. However, their precise natures and molecular mechanisms have not been elucidated. In this study, we studied TIDs recruited in progressive P815 and regressive P198 tumors of the same origin. Our data showed that P815 tumors contained CD4+ 8+ and CD4- 8- TID815 subsets, whereas P198 tumors contained CD4+ 8+ and CD4+ 8- TID198 subsets. They similarly stimulate allogeneic T cell proliferation and have nitric oxide-mediated cytotoxicity to tumor cells with an exception of CD4- 8- TID815 with less efficiency. The newly identified fourth CD4+ 8+ TID815 or TID198 subset and the CD4+ 8- TID198 all express high levels of IFN-gamma and interleukin (IL)-6, whereas CD4- 8- TID815 secrete a marked level of transforming growth factor-beta. Vaccination of mice with P815 tumor lysate-pulsed CD4+ 8+ TID815 or TID198 and CD4+ 8- TID198 induced IFN-gamma-secreting Th1 and effective CTL responses leading to protective immunity against P815 tumor, whereas CD4- 8- TID815 stimulated IL-10-expressing Tr1 responses leading to immune suppression. Transfer of CD4+ Tr1 cells obtained from CD4- 8- TID815-immunized wild-type, but not IL-10(-/-) mice, into CD4+ 8+ TID815 immunized mice abolished otherwise inevitable development of antitumor immunity. Taken together, our findings provide an important insight into immunologic alterations in progressive and regressive tumors and an implication for dendritic cell-based approaches in the design of cancer vaccines.     

Differential expression of and responsiveness to transforming growth factor-beta (TGF-beta) isoforms in hormone-dependent and independent lines of mouse mammary tumors.

Transforming growth factor-beta2 (TGF-beta2) and -beta3 mRNA expressions were studied in ductal hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. MPA treatment of HD tumors induced a significant decrease in TGF-beta2 and -beta3 mRNA levels. Progression to an HI phenotype of ductal tumors was associated with reduced TGF-beta2 and -beta3 expressions, as compared with their HD counterparts. Exogenously added TGF-beta1, -beta2, and -beta3 (1 ng/ml) inhibited the proliferation of primary cultures of epithelial cells from ductal HD and HI tumors. In addition, TGF-beta expression and effects were studied in the other type of MPA-induced mammary tumors, which are of lobular origin and lack steroid hormone receptors and evidence an HI behavior. These lobular HI lines showed TGF-beta2 levels similar to those found in HD lines growing in MPA-treated mice. In contrast, TGF-beta3 mRNA levels were 12- to 20-fold higher than in HD tumors. Primary cultures of lobular HI epithelial cells required either TGF-beta concentrations of 10 ng/ml to show an inhibitory response, or were completely resistant to TGF-beta inhibition. Studies of the molecular mechanisms involved in reduction or loss of TGF-beta responsiveness in lobular HI tumors showed that cell surface type II TGF-beta receptor levels were lower in these tumors than those present in HD tumors. Our results support the hypothesis that TGF-beta could play a role as an autocrine growth inhibitor in HD and HI ductal tumors. Autonomous growth of lobular HI tumors could be favored by undetectable or low TGF-beta1 and -beta2 expressions and by reduced or lost sensitivity of epithelial cells to TGF-beta's antiproliferative effects. However, the extremely high levels of TGF-beta3 expression in lobular HI tumors, in spite of reduced sensitivity to TGF-beta3 inhibitory growth effect in tumor epithelial cells, suggest a net positive role for TGF-beta3 in these tumors.     

Correlation between reduced p21(WAF1/CIP1) expression and abnormal p53 expression in esophageal carcinomas

Direct gene transfer into somatic tissue iii vivo is a developing technology with potential application for cancer gene therapy. In this study, recombinant vaccinia virus encoding human IL-2 gene (rVV-IL-2) was used as a candidate vector in mediating iii vivo gene therapy. After rVV-IL-2 was expanded in VERO cells for 72 h, high titer (10(8)-10(10) PFU/ml) rVV-IL-2 were harvested. When 10(6) murine melanoma cells (F16-F10) were infected with rVV-IL-2, about 200 U/ml IL-2 activity was detected in the supernatants at 8 h, and the up-regulation of ICAM-1 and MHC-I expressions on the melanoma cells were observed. The treatment of murine melanoma model by local injection of rVV-IL-2 into the tumor site showed that rVV-IL-2 transfection significantly inhibited the tumor growth and prolonged the survival time of tumor-bearing mice. The splenocytes from rVV-IL-2 treated mice showed higher cytotoxicities of NK, LAK and CTL in comparison with those from the controls. These results suggest that in vivo transfection mediated by rVV-IL-2 has potential effectiveness in enhancing host immunity and would be a useful approach to cancer gene therapy.     

Vaccination with viable human umbilical vein endothelial cells prevents metastatic tumors by attack on tumor vasculature with both cellular and humoral immunity.

PURPOSE: Because tumor endothelium is rarely targeted by immunity but is critically important for tumor growth, the immunity against tumor endothelium is to be developed as a novel antitumor strategy. EXPERIMENTAL DESIGN: First, viable human umbilical vein endothelial cells (HUVEC) were immunized to C57BL/6 and BALB/c mice to evoke specific CTLs as well as antibodies against tumor endothelium. Lewis lung carcinoma or myeloma cells were subsequently inoculated to evaluate the effect on tumor growth by vaccination. Second, the effect on tumor metastasis by vaccination was studied using tumor-resected mice receiving HUVEC immunization 3 days after excision. Third, the immune sera and T lymphocytes from HUVEC-immunized mice were transferred to tumor-bearing mice and added to cultured HUVECs to investigate their antiproliferative effect. RESULTS: Viable HUVEC immunization showed potent antitumor effects in Lewis lung carcinoma and myeloma tumor models. Both immune sera and CTL inhibited tumor growth and specifically suppressed proliferation of HUVECs. Particularly, tumors entirely disappeared on day 90 after tumor inoculation in four of six tumor-bearing mice receiving CTL therapy. In a metastatic tumor model, we found that the HUVEC vaccination prolonged life span from 30.9 to 41.5 days after tumor resection compared with PBS-treated mice without apparent side effects. CONCLUSIONS: Vaccination with viable HUVECs evoked both humoral and cellular immunity against tumor microvasculature, and therefore significantly inhibited tumor growth and prolonged life span of tumor-resected mice. This may provide with a novel treatment for metastatic tumors. Moreover, we have established a convenient method to evoke specific CTL against tumor angiogenesis.     

CS-706, a novel cyclooxygenase-2 selective inhibitor, prolonged the survival of tumor-bearing mice when treated alone or in combination with anti-tumor chemotherapeutic agents

The potent chemopreventive activity of cyclooxygenase-2 (COX-2) inhibitors has been demonstrated in a number of preclinical studies, but their potency in antitumor activity is still in dispute. In this report, we demonstrate the potent antitumor activity of a novel COX-2 inhibitor, CS-706 in mouse colorectal adenocarcinoma colon 26 tumor-bearing mice treated with or without antitumor chemotherapeutic agents. Daily oral administration of CS-706 at doses of 3-100 mg/kg from the day of tumor inoculation (Day 0) inhibited tumor growth dose-dependently, and the maximal inhibition was 67% at a dose of 100 mg/kg. In contrast, celecoxib, a well-known COX-2 inhibitor, did not inhibit tumor growth at doses up to 100 mg/kg. Furthermore, CS-706 at a dose of 1 mg/kg or above markedly prolonged the survival time of tumor-bearing mice. Administration of 30 mg/kg CS-706 from Day 7 combined with a single intravenous treatment of 10 mg/kg cisplatin on Day 7 completely regressed the tumors in all tumor-bearing mice examined, whereas only in 1 of 10 mice tumor was regressed with cisplatin treatment. Similar combination effects were observed with 10 mg/kg CS-706 and 60 mg/kg 5-fluorouracil (5-FU). Moreover, 10 mg/kg CS-706 significantly inhibited angiogenesis induced by implanted chambers with colon 26 cells in a dorsal air sac assay in mice. Collectively, these results suggest that CS-706 is a potent antitumor agent, especially in combination with conventional chemotherapeutic agents, and that the anti-angiogenic activity of CS-706 may contribute at least in part to its marked antitumor activity.     

Improved efficacy of dendritic cell vaccines and successful immunization with tumor antigen peptide‐pulsed peripheral blood mononuclear cells by coadministration of recombinant murine interleukin‐12

The well‐characterized P815 tumor model was used to optimize anti‐tumor immunization approaches in mice. Tumor peptides derived from antigens P198 or P1A were targeted to antigen‐presenting cells (APC) by ex vivo pulsing. Initial experiments with irradiated pulsed splenic dendritic cells (sDC) injected weekly in the hind footpads for 3 weeks demonstrated cytolytic T lymphocyte (CTL) generation in 10–20% of mice. Because of the importance of interleukin‐12 (IL‐12) in tumor rejection responses, pulsed sDCs also were given together with recombinant murine IL‐12 (rmIL‐12). This strategy induced peptide‐specific CTL in 100% of the mice. The IL‐12 had to be injected in the footpads on days 0, 1 and 2 of each immunization week to achieve an optimal effect. The improvement seen with the addition of IL‐12 prompted examination of other sources of APC. Purified resting B cells, lipopolysaccharide (LPS) blasts and non‐fractionated splenocytes or peripheral blood mononuclear cells (PBMC) were pulsed with peptide and administered with the same schedule of rmIL‐12. Because these cell types appeared to bind peptides less avidly than did DC, increasing peptide doses were used during pulsing. Interestingly, immunization with each of these APC also induced specific CTL in 100% of mice, provided rmIL‐12 was coadministered. CTLs were detected both in the spleen and in the peripheral blood. Immunization with irradiated, P1A‐pulsed PBMC plus rmIL‐12 resulted in protection against challenge with tumors expressing the specific antigen in all mice. The ease by which human patient PBMCs can be prepared provides a straightforward vaccination approach to be used in clinical trials of peptide‐based immunization in melanoma. Int. J. Cancer 80:324–333, 1999. © 1999 Wiley‐Liss, Inc.     

Cytokine production of lung cancer cell lines: Correlation between their production and the inflammatory/immunological responses both in vivo and in vitro

Cytokines produced by tumor cells may have various effects on antitumor immune responses and tumor growth. In the present study, the cytokine production of 31 lung cancer cell lines was evaluated, while any correlation with the histological type, the induction of tumor-specific cytotoxic T lymphocytes (CTL) in vitro, and angiogenesis and the infiltration of inflammatory cells in tumor tissues were also examined. Production of interleukin (IL)-1alpha, IL-1beta, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor, transforming growth factor (TGF)-beta and vascular endothelial growth factor (VEGF) in the culture supernatant was measured using enzyme-linked immunosorbent assay. Each cytokine was produced in a substantial number of the tumor cell lines. In particular, IL-6, IL-8, TGF-beta and VEGF were produced in 18 (55%), 29 (94%), 31 (100%) and 28 (90%) of 31 cell lines, respectively. However, neither IL-4 nor TNF-alpha was produced at all by any tumor cell line. TGF-beta production was significantly higher in adenocarcinoma than in squamous cell carcinoma (P = 0.03). Immunohistochemical staining revealed the magnitude of macrophage infiltration, and angiogenesis in surgically resected tumor tissue specimens correlated well with GM-CSF and IL-8 production from the corresponding cell lines. Among six lung cancer cell lines, CTL were induced in the three lung cancer cell lines that produced a lower amount of TGF-beta (<100 pg/mL). These findings suggested that TGF-beta produced by tumor cells could inhibit the induction of CTL in vitro. The present results suggest that the production of various cytokines from tumor cells might exert various paracrine effects both in vivo and in vitro.     

Role of interleukin 2 and a serum suppressive factor on the induction of activated killer cells cytotoxic for autologous human melanoma cells

Activated killer (AK) cells cytotoxic for freshly prepared autologous melanoma cells were generated in vitro when recombinant interleukin 2 (rIL2) was incubated with peripheral blood mononuclear cells from 11 of 12 patients with metastatic melanoma. The cytotoxic response first appeared significant after 3 days of culture with rIL2; it peaked after 6 days and then declined after 9 days of incubation. Peripheral blood mononuclear cells cultured with medium alone (control) or with recombinant gamma-interferon alone (200 units/ml) failed to acquire AK cytotoxicity at any time in any of the 12 patients. Autologous tumor cells also could not induce AK activity in mixed-lymphocyte tumor cell cultures. The level of AK activity generated was significantly reduced (P less than 0.001) when 10% autologous human serum was used instead of 10% fetal calf serum in the rIL2 cultures. This suppression was specific for the inductive phase of AK activity, because autologous human serum could not suppress the cytotoxic capability of AK cells once they were activated by rIL2. The serum suppressive effect on the induction of AK cytotoxicity could be overcome by increasing the dose of rIL2 or by adding recombinant gamma-interferon to the cultures. Lymphocytes from melanoma patients thus have the latent ability to kill autologous melanoma tumor cells. However, this cytotoxic capability requires an interleukin 2-induced differentiation step that could be amplified by gamma-interferon and inhibited by a serum suppressive factor.