Sensitizing T-lymphocytes for adoptive immunotherapy by vaccination with wild-type or cytokine gene-transduced melanoma

Background: For the relatively nonimmunogenic B16-F10 murine melanoma, it has been found that genetically engineered expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin (IL)-2, IL-4, or interferon-gamma (IFN-) resulted in a vaccine that could induce resistance to rechallenge. Because T cells from lymph nodes draining the sites of some progressive tumors can mediate tumor regression after in vitro activation, it seemed possible that even apparently nonimmunogenic melanoma cells might induce similar preeffector cells in the vaccine-draining lymph nodes (DLNs). Methods: C57BL/6 mice were vaccinated with B16-F10 cells that were either unmodified or genetically modified to produce IL-2, IL-4, GM-CSF, or IFN-. DLNs were harvested 10 days after vaccination for adoptive immunotherapy (AIT). The DLN cells were activated with bryostatin 1 and ionomycin (B/I), expanded for 10 days in culture, and transferred to mice with 3-day pulmonary metastases. Pulmonary nodules were counted 14 days after AIT. Results: Adoptive transfer of expanded DLN lymphocytes sensitized by inoculation of WT B16-F10, or IL-4, GM-CSF, or IFN- expressing cells significantly reduced pulmonary metastases. Despite the spontaneous regression of IL-2-transduced B16-F10 tumors, DLN from mice inoculated with IL-2 producing B16 cells had little or no antitumor activity. Conclusions: B16-F10 vaccination strategies that apparently do not induce systemic immunity can effectively sensitize DLN preeffector cells.Presented at the 49th Annual Cancer Symposium of The Society of Surgical Oncology, Atlanta, Georgia, March 21–24, 1996.     

Immunogene therapy with interleukin-2-secreting fibroblasts for intracerebrally metastasizing breast cancer in mice

OBJECT: Breast cancer is the second leading cause of cancer-related death in American women. Brain metastases occur in 15 to 30% of patients with breast cancer, and this usually results in death. Despite the availability of surgery, radiotherapy, and chemotherapy, the prognosis for patients with breast cancer that has metastasized to the intracerebral region remains poor. This study was designed to determine if an intracerebrally metastasizing breast tumor could be treated successfully with a cellular vaccine consisting of allogeneic fibroblasts (H-2K) modified to secrete interleukin (IL)-2. METHODS: The authors used EO771 breast cancer cells, derived from a spontaneously arising breast-cancer tumor in C57BL/6 mice. The authors first determined the length of survival of C57BL/6 mice that had been injected with varying numbers of EO771 cells into the right frontal lobes and found that 100% of those animals that received a dose of 10(4) cells died within 41 days, whereas 100% of the group that received 10(3) cells died within 23 days. Based on these results, 5 x 10(4) EO771 cells were injected into the right frontal lobe of C57BL/6 mice and the animals were treated intracerebrally with a single intratumoral injection of 10(6) allogeneic fibroblasts genetically engineered to secrete IL-2. The allogeneic fibroblasts transfected with the IL-2 gene formed large quantities of IL-2 as measured by an enzyme-linked immunosorbent assay. Control groups of animals were treated with either allogeneic fibroblasts transfected with the retroviral vector, but not the IL-2 gene, or with media. The effects of this treatment on the animal's survival and the tumor's histopathological characteristics were investigated. The results indicate a significant prolongation (p < 0.005) of survival in animals with intracerebrally metastasizing breast cancer treated only with IL-2-secreting allogeneic fibroblasts. Tumor did not develop in four of 10 animals in the IL-2-treated group, and these animals were rechallenged at 90 days by intracerebral injection of tumor, but no treatment cells, and compared with four naive animals receiving intracerebral tumor. Again, animals that previously had been treated with IL-2-secreting fibroblasts had a markedly prolonged survival (p < 0.05) compared with control animals following a second challenge with tumor cells. Histopathological examination revealed smaller tumors associated with lymphocytic infiltrations in the treated animals. CONCLUSIONS: This work represents a new treatment for breast cancer that has metastasized to the brain in which a cellular vaccine consisting of allogeneic fibroblasts genetically engineered to secrete cytokines is used as a novel means for delivery of immunogene therapy and demonstrates the induction of long-term immunity against tumor.     

Cytokines as an adjuvant to tumor vaccines: Efficacy of local methods of delivery

Background: We examined alternative methods of delivering cytokines as an adjunct for priming lymph node (LN) cells draining sites of vaccine inoculation for the purpose of generating immune cells for adoptive immunotherapy. Methods: Using syngeneic murine tumors we examined the ability of IL-2, IL-4, or GM-CSF delivered locally to a site of tumor inoculum to induce antitumor reactive draining LN cells. Mice were inoculated subcutaneously with tumor cells transduced to secrete cytokine; tumor cells admixed with fibroblasts transduced to secrete cytokine; or intralesional inoculation of cytokine in established tumor to induce sensitized LN cells capable of mediating tumor regression in adoptive transfer. Results: Both IL-4 and GM-CSF cytokines were effective in enhancing the antitumor reactivity of vaccine-primed LN cells compared to IL-2, which was ineffective. The local delivery of GM-CSF by autocrine or paracrine secretion of genetically engineered cells, as well as direct intratumoral delivery was capable of upregulating LN sensitization compared to systemic administration, which did not. Conclusions: The local delivery of GM-CSF as an adjuvant for tumor vaccination can be accomplished by various methods, including direct injection, which avoids the need for gene transfer.     

Adjuvant effect of a Flt3 ligand (FL) gene-transduced xenogeneic cell line in a murine colon cancer model

BACKGROUND: Flt3 Ligand (FL) has been shown to elicit antitumor responses induced by tumor antigen stimulation. Allogeneic and xenogeneic cell lines transduced with cytokine genes may be used to augment the antitumor efficacy of tumor antigens. OBJECTIVES: The objective was to evaluate the augmentation of tumor lysate-induced immunity by a more clinically applicable FL gene-transduced xenogeneic cell line in combination with interleukin-2 (IL-2) in a CC-36 murine colon cancer model. METHODS: Human 143B osteosarcoma tumor cells were transduced with full-length FL cDNA (143B-FL). Secretion of FL from 143B-FL was analyzed in vivo in normal BALB/c mice transplanted with 143B-FL, and expansion of dendritic cells (DC) was also analyzed in the same mice by flow cytometry. Eight-week-old, male BALB/c mice were used in a prophylactic vaccination protocol utilizing tumor lysate (CLy), 143B-FL, and soluble IL-2. Prophylactic group designations (n = 10/group) were as follows: ten million 143B-FL cells (alone, with tumor lysate, or with tumor lysate and IL-2), IL-2 with tumor lysate, IL-2 alone, or a no treatment control. The tumor lysate (200 microg of protein) and IL-2 (100,000 IU) injections were administered intraperitoneally. Mice were challenged subcutaneously with 10(3) CC-36 tumor cells. Tumor protection and tumor burden (TB), as mean tumor diameter, were determined. Peripheral blood lymphocytes (PBLs) from the 143B-FL + IL-2 + tumor lysate vaccinated group were analyzed for cytolytic activity in 4-h chromium release assays. In addition, plasma cytokine concentrations of interleukin-12 (IL-12) and interferon gamma (IFN-gamma) were assessed by ELISA. Student's t tests were used for all statistical comparisons. RESULTS: In vivo expression of FL was observed 24 h following the inoculation of 143B-FL, and a four fold increase in DCs was observed in the peripheral blood of these mice. Mice immunized with a combination of 143B-FL, tumor lysate and IL-2 showed statistically significant protection against tumor development (10%) for 100 days after tumor challenge; incidences in other groups ranged from 40 to 100% (P < 0.05). Moreover, this immunization protocol produced the lowest TB at 3- and 6-week time points (0, 1.6 mm) when compared to all other groups (TB between 7.2 and 15.9 mm) (P < 0.05). In addition, PBLs from vaccinated mice showed increased cytolytic activity against CC-36 target cells. This corresponded to increased levels of IL-12 and IFN-g in the plasma of mice following vaccination. CONCLUSIONS: These data suggest that FL gene-transduced xenogeneic tumor cells may augment the immunity induced by tumor antigens and systemic IL-2 through the activation of dendritic cells and T-cell-mediated mechanisms.     

Administration of interleukin-12 and -18 enhancing the antitumor immunity of genetically modified dendritic cells that had been pulsed with Semliki forest virus-mediated tumor complementary DNA

OBJECT: Immunogene therapy for malignant gliomas was further investigated in this study to improve its therapeutic efficacy. METHODS: Dendritic cells (DCs) were isolated from bone marrow and pulsed with phosphate-buffered saline or Semliki Forest virus (SFV)-mediated 203 glioma complementary (c)DNA with or without systemic administration of interleukin (IL)-12 and IL-18 to treat mice bearing the 203 glioma. To study the immune mechanisms involved in tumor regression, the authors investigated tumor growth of an implanted 203 glioma model in T cell subset-depleted mice and in interferon (IFN) gamma-neutralized mice. To examine the protective immunity produced by tumor inoculation, a repeated challenge of 203 glioma cells was given by injecting the cells into the left thighs of surviving mice and the growth of these cells was monitored. The authors demonstrated that the combined administration of SFV-cDNA, IL-12, and IL-18 produced significant antitumor effects against the growth of murine glioma cells in vivo and also can induce specific antitumor immunity. The synergic effects of the combination of SFV-cDNA, IL-12, and IL-18 in vivo were also observed to coincide with markedly augmented IFN-gamma production. The antitumor effects of this combined therapy are mediated by CD4+ and CD8+ T cells and by NK cells. These results indicate that the use of IL-18 and IL-12 in DC-based immunotherapy for malignant glioma is beneficial. CONCLUSIONS: Immunogene therapy combined with DC therapy, IL-12, and IL-18 may be an excellent candidate in the development of a new treatment protocol. The self-replicating SFV system may therefore provide a novel approach for the treatment of malignant gliomas.     

A novel dendritic cell-based cancer vaccine produces promising results in a syngenic CC-36 murine colon adenocarcinoma model

BACKGROUND: This study was conducted to test the efficacy of a new cancer vaccine, composed of dendritic cells (DCs) pulsed with an interleukin-2 gene-encoded vaccinia virus tumor oncolysate (DC-IL-2VCO) in a CC-36 murine colon adenocarcinoma model. MATERIALS AND METHODS: CC-36 tumor cells were injected subcutaneously into the left flank of four- to six-week old male BALB/c mice. The mice were divided into three groups, each of which received one of the following treatments: (1) DCs pulsed with the IL-2 gene-encoded vaccinia oncolysate (DC-IL-2VCO), (2) DCs pulsed with the tumor oncolysate alone (DC-CO), or (3) no treatment (control). Tumor incidence was measured, and survival rates were compared using a paired Student's t-test. Cytolytic T cell activity was measured in peripheral blood lymphocytes (PBL) and splenic lymphocytes using a (51)Cr-release assay. Lastly, mice were depleted of either CD4+ or CD8+ lymphocytes prior to receiving the vaccine to test the mechanism of tumor immunity in these mice. RESULTS: Mice treated with DC-IL-2VCO demonstrated decreased tumor burden, increased survival, and greater cytolytic activity compared with control mice and mice receiving DC-CO. In addition, mice depleted of CD8+ T cells prior to immunization with IL-2VV + DC-IL-2VCO had a significant increase in the incidence of tumor, similar to the untreated control mice. CONCLUSIONS: DCs pulsed with an IL-2 gene-encoded vaccinia virus tumor oncolysate (DC-IL-2VCO) produced safe and effective immune responses in a murine CC-36 colon adenocarcinoma model. This vaccine (DC-MelVac; Patent no. 11221/5) has the potential to treat humans with cancer, and has received FDA approval for use in Phase I clinical trials.     

Local secretion of IL-12 augments the therapeutic impact of dendritic cell–tumor cell fusion vaccination

Background

The development of dendritic cell (DC)–tumor fusion vaccines is a promising approach in cancer immunotherapy. Using fusion vaccines allows a broad spectrum of known and unidentified tumor-associated antigens to be presented in the context of MHC class I and class II molecules, with potent co-stimulation provided by the DCs. Although DC–tumor fusion cells are immunogenic, murine studies have shown that effective immunotherapy requires a third signal, which can be provided by exogenous interleukin 12 (IL-12). Unfortunately, systemic administration of IL-12 induces severe toxicity in cancer patients, potentially precluding clinical use of this cytokine to augment fusion vaccine efficacy. To overcome this limitation, we developed a novel approach in which DC–tumor fusion cells locally secrete IL-12, then evaluated the effectiveness of this approach in a murine B16 melanoma model.

Materials and methods

Tumor cells were stably transduced to secrete murine IL-12p70. These tumor cells were then electrofused to DC to form DC–tumor heterokaryons. These cells were used to treat established B16 pulmonary metastases. Enumeration of these metastases was performed and compared between experimental groups using Wilcoxon rank sum test. Interferon γ enzyme-linked immunosorbent spot assay was performed on splenocytes from treated mice.

Results

We show that vaccination with DCs fused to syngeneic melanoma cells that stably express murine IL-12p70 significantly reduces counts of established lung metastases in treated animals when compared with DC–tumor alone (P = 0.029). Interferon γ enzyme-linked immunosorbent spot assays suggest that this antitumor response is mediated by CD4⁺ T cells, in the absence of a tumor-specific CD8⁺ T cell response, and that the concomitant induction of antitumor CD4⁺ and CD8⁺ T cell responses required exogenous IL-12.

Conclusions

This study is, to the best of our knowledge, the first report that investigates the impact of local secretion of IL-12 on antitumor immunity induced by a DC–tumor fusion cell vaccine in a melanoma model and may aid the rational design of future clinical trials.     

Potent antitumor effects of CD154 transduced tumor cells in experimental bladder cancer

PURPOSE: Current intravesical immunotherapy for bladder cancer with bacillus Calmette-Guerin instillations is standard treatment for patients with high risk superficial tumors but relapses are common. We evaluated the tumor vaccine concept in murine bladder cancer by comparing tumor cell transduction with genes coding for the immunostimulatory molecules CD154, interleukin (IL)-12 and CD80 to design a novel vaccination strategy. MATERIALS AND METHODS: Adenoviral vectors were used to transduce murine bladder cancer MB-49 cells with genes coding for CD154, IL-12 and CD80. Parental or transduced MB-49 cells were injected subcutaneously into syngeneic mice. The effects of transgene expression on tumorigenicity and the generation of protective immunological memory against challenge with parental tumor were studied. RESULTS: All 76 animals injected with parental MB-49 cells had tumors within 8 to 12 days. Tumor cell expression of CD154 combined with IL-12 completely inhibited tumor outgrowth with all 21 mice tumor-free and CD154 transduction alone was almost as effective with 33 of 35 tumor-free. IL-12 production by tumor cells delayed tumor outgrowth and 4 of 10 mice remained tumor-free. Over expression of CD80 had no effect on tumorigenicity. CD154 expressing tumors were rapidly infiltrated with large numbers of CD4+ and CD8+ T cells. Mice vaccinated 4 times with adenoviral CD154 transduced MB-49 cells were completely protected against challenge with parental tumor. Co-injection of CD154 modified cells with parental MB-49 cells retarded tumor growth. CONCLUSIONS: Our experimental results suggest that the potent antitumor effects of CD154 gene transduction should be considered for immunostimulatory gene therapy for bladder cancer.     

Antitumor activity of interleukin-12 against murine bladder cancer

PURPOSE: We investigated the antitumor activity of interleukin-12 (IL-12) against MBT-2, a murine bladder carcinoma, to clarify whether or not IL-12 is effective against urothelial tumors. MATERIALS AND METHODS: MBT-2, a murine carcinogen-induced, poorly differentiated transitional cell carcinoma of C3H/He origin, was used. Three or 10 days after the subcutaneous administration of MBT-2 cells, C3H/He mice were injected intraperitoneally with IL-12 five times per wk. for 2 wk. Tumor growth was measured twice weekly. Spleen cells from the C3H/He mice that had rejected MBT-2 after the IL-12 treatment were examined for MBT-2-specific cytolytic T lymphocytes (CTL) activity and cytokine production. RESULTS: Tumor growth and acceptance was obviously suppressed when C3H/He mice were treated with IL-12 from 3 days after the tumor inoculation. In the spleen cells from the C3H/He mice that had rejected MBT-2, MBT-2-specific CTL activity and secretion of IL-2 and interferon (IFN)-gamma were clearly detected. However, the established MBT-2 tumor cells were not rejected when C3H/He mice were given IL-12 from 10 days after the tumor inoculation, although the tumor growth was transiently suppressed during the IL-12 treatment. CONCLUSION: These data demonstrate that IL-12 is considerably effective against murine bladder cancer and suggest the clinical application of IL-12 against human bladder cancer.     

自体肿瘤疫苗治疗肝癌的实验研究

目的探讨自体肿瘤疫苗预防和治疗肝癌的效果及抗癌机制。方法采用多聚甲醛固定的小鼠Hepal-6细胞为抗原、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白介素-2（IL-2）缓释微球为免疫激活剂、辅以免疫辅助剂TiterMax Gold的肝癌疫苗皮内接种C57BL／6J小鼠，观察肿瘤疫苗预防和治疗肝癌的效果。结果对照组15只小鼠全部发展成肝肿瘤；含有固定Hepal6细胞和IL-2及GM—CSF微球的肿瘤疫苗，80％小鼠获得保护。再加入免疫辅助剂TiterMax Gold的肿瘤疫苗，则87％小鼠获得保护。将Hepal6细胞接种于左躯干皮下。肿瘤长至直径5mm时，皮内接种肿瘤疫苗2次。结果显示，对照组肿瘤继续生长。疫苗组在第2次接种后7～10d，肿瘤生长受到抑制，随后明显缩小。60％小鼠的肿瘤完全消失。细胞毒性实验结果显示，接种疫苗的小鼠脾细胞的杀瘤活性可被抗-CD3^＋、抗-CD8^＋、抗-MHC-Ⅰ单克隆抗体所阻断，但不被抗-CD4^＋、抗-MHC-Ⅱ单克隆抗体所阻断。结论自体肿瘤疫苗具有很强的抗肿瘤的效果。其抗瘤机制是诱导内源性抗原特异性CTL反应，由典型的MHC—Ⅰ限制的CD8^＋T细胞所介导的。     

Inhibition of Host Signal Transducer and Activator of Transcription Factor 6 Results in Cure With Cyclophosphamide and Interleukin 12 Immunotherapy

BACKGROUND: Interleukin (IL)-12 immunotherapy is highly effective against established immunogenic tumors. However, nonimmunogenic tumors fail to respond to IL-12 therapy. Analysis of tumor rejection of the immunogenic tumors shows that a preexisting antitumor immune response is required for an effective IL-12 response. It is not known whether this lack of a preexisting host antitumor immune response is a limiting factor for the lack of response to IL-12 therapy by nonimmunogenic tumors. METHODS: Experiments were done using the spontaneously arising nonimmunogenic metastatic murine breast 4T1 carcinoma in normal and STAT6 knockout BALB/c mice. RESULTS: 4T1 is nonimmunogenic in normal mice, and established subcutaneous tumors are resistant to immunotherapy with cyclophosphamide (Cy) plus IL-12. However, in STAT6 knockout mice, 4T1 becomes immunogenic, and established 4T1 tumors are eradicated by Cy plus IL-12. Adoptive transfer of spleen cells from normal mice into STAT6 knockout mice before tumor inoculation reduces both the immunogenicity and response to Cy plus IL-12 immunotherapy of 4T1 in the recipient mice. CONCLUSIONS: Cy plus IL-12 immunotherapy can eradicate nonimmunogenic tumors as long as a preexisting immunity is established in the tumor-bearing host. Furthermore, the STAT6 pathway is likely involved in the suppression of the development of host antitumor immunity.     

Addition of interleukin-12 to GA733 tumor protein vaccine leads to development of tumor protective immunity despite surgical stress

Background: We have previously shown that preoperative vaccination with the GA733 protein does not inhibit tumor growth in mice undergoing open surgery or carbon dioxide insufflation. In this study we assessed the antitumor effect of a combined GA733 and interleukin-12 (IL-12) vaccine. Methods: For this study, BALB/c mice were immunized with GA733, IL-12, or GA733 and IL-12, or they received no vaccine. Immediately after surgery (laparotomy or insufflation), GA733-transfected CT26 cells (C26-GA733) were injected subcutaneously into all mice. After 5 weeks, the mice were sacrificed, their tumors measured, GA733-specific antibodies determined by enzyme-linked immunoassay, and GA-733-specific cytotoxicity tested by flow cytometry using labeled C26-GA733 cells. Results: Tumors were significantly (p < 0.05) smaller in both the insufflation and open groups that received combined GA733 and IL-12 than in their respective control subjects. Vaccination also induced a significant increase in the antibody and cell-mediated tumor-specific immunity. Conclusion: A preoperative vaccine consisting of GA733 and IL-12 inhibited postoperative tumor growth after open and closed surgery and allowed the mice to overcome the immunosuppressive effects of surgery.     

Induction of an antitumor immunological response by an intratumoral injection of dendritic cells pulsed with genetically engineered Semliki Forest virus to produce interleukin-18 combined with the systemic administration of interleukin-12

OBJECT: The aim of this study was to investigate further immunogene treatment of malignant brain tumor to improve its therapeutic efficacy. METHODS: Intratumoral dendritic cells pulsed with Semliki Forest virus (SFV)-interleukin-18 (IL-18) and/or systemic IL-12 were injected into mice bearing the B16 brain tumor. To study the immune mechanisms involved in tumor regression, we monitored the growth of implanted B16 brain tumor cells in T cell-depleted mice and IFNgamma-neutralized mice. To analyze the protective immunity created by tumor inoculation, B16 cells were injected into the left thighs of mice that had received an inoculation, and tumor growth was monitored. The local delivery of dendritic cells pulsed with IL-18 bound by SFV combined with the systemic administration of IL-12 enhanced the induction of the T helper type 1 response from tumor-specific CD4+ and CD8+ T cells and natural killer cells as well as antitumor immunity. Interferon-gamma is partly responsible for this IL-18-mediated antitumor immunity. Furthermore, the protective immunity is mediated mainly by CD8+ T cells. CONCLUSIONS: Immunogene therapy that combines the local administration of dendritic cells pulsed with IL-18 bound by SFV and the systemic administration of IL-12 may be an excellent candidate for the development of a new treatment protocol. A self-replicating SFV system may therefore open a novel approach for the treatment of malignant brain tumor.     

辅助性T细胞和细胞毒性T淋巴细胞双表位修饰的树突状细胞肿瘤疫苗治疗胃癌的实验研究

目的研究辅助性T细胞（Th）表位和细胞毒性T淋巴细胞（CTL）双表位修饰的树突状细胞（DCs）肿瘤疫苗用于胃癌免疫治疗的效果。方法用CTL表位MAGE-341-49和Th表位MAGE-322-36混合多肽冲击DCs，每周刺激脾脏T细胞1次，4周后收集多肽特异性T细胞。流式仪分析T细胞亚群分布，测定CD4^＋T细胞识别抗原细胞因子分泌及CD8^＋T细胞杀伤肿瘤细胞效能，观察双表位修饰的DCs肿瘤疫苗治疗胃癌的保护性免疫效应。结果双表位致敏的DCs体外可同时活化CD4^＋和CD8^＋T细胞，其中CD4^＋T细胞识别肿瘤细胞小鼠前胃癌细胞株MFC后分泌大量Th1型细胞因子[干扰素（IFN）-γ，白介素（IL）-2]，CD8^＋T细胞强效杀伤MFC。双表位修饰的DCs肿瘤疫苗小鼠体内免疫治疗获得抵抗后继胃癌细胞MFC的免疫保护能力，并显著高于单一表位（CTL或Th）修饰的DC8疫苗。结论Th和CTL双表位修饰的DCs肿瘤疫苗可同时激活CD4^＋Th1细胞和CD8^＋CTL抗肿瘤免疫，有效清除胃癌细胞。     

Photodynamic therapy and anti-tumor immunity

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) efficacy appears to be enhanced in the presence of an intact immune system and PDT has been shown to augment anti-tumor immunity. The mechanisms leading to the enhancement of the host immune response to tumor are unclear. Anti-tumor immunity depends upon the presence of activated antigen presenting cells (APCs). These cells are activated by their recognition of components released by pathogens, viruses, dead cells, and the presence of pro-inflammatory mediators. Activated APCs stimulate the generation of cytokine secreting effector cells. Therefore, we have hypothesized that PDT generated inflammatory mediators and components released from tumor cells killed by PDT results in the activation of APCs capable of stimulating effector T-cell proliferation and cytokine secretion. STUDY DESIGN/MATERIALS AND METHODS: To determine the effect of PDT on APCs, tumor draining lymph nodes (TDLNs) of EMT6 or Colo 26 tumor bearing mice were isolated 24 hours after Photofrin-PDT and flow cytometry was used to detect the presence of APCs secreting the T cells stimulatory cytokine, IL-12. APCs were also isolated from TDLNs and used to stimulate T-cell proliferation and secretion of interferon-gamma (IFN-gamma). RESULTS: PDT results in an increase in IL-12 expressing APCs in the TDLN. This increase was accompanied by an increase in the ability of APCs isolated from TDLNs of PDT-treated mice to stimulate T-cell proliferation and T-cell secretion of IFN-gamma. CONCLUSIONS: Our results indicate that APCs isolated from PDT-treated mice exhibit an enhanced ability to stimulate T-cell proliferation and IFN-gamma secretion, suggesting that PDT results in increased APC activity.     

Dendritic cells (DC) loaded with apoptotic, allogeneic melanoma cells reduce tumor progression and improve survival in a murine immunotherapy model

Introduction: DC loaded with apoptotic, syngeneic melanoma cells slow tumor growth and improve survival in a murine immunotherapy model. The use of allogeneic cells as a source of tumor antigen could increase the clinical applicability of this therapy, so we investigated the efficacy of DC loaded with apoptotic, allogeneic cells in this model. Methods: DC were cultured from bone marrow from C-57/BL-6 mice with GMCSF (10 ng/ml), IL-4 (5 ng/ml). Syngeneic B-16 or allogeneic K-1735 melanoma cells were irradiated (20 Gy) to induce apoptosis and cultured with DC at a 1:1 ratio for 48 hr to make DC/Apo vaccine. C-57/BL-6 mice (n = 6) were injected with 2.5 x 10⁵ B-16 SQ and treated with 1 x 10⁶ DC/Apo vaccine cells SQ or saline control weekly x4 starting 5 days after tumor implantation. In other experiments, splenocytes were harvested from control and immunized tumor-bearing animals at day 30 and stimulated weekly x2 with IFN-gamma treated, irradiated B-16 cells. Cytotoxic T lymphocyte (CTL) assays were performed with B-16 targets for 48 hr and % specific lysis calculated. Results: Summarized in table 1. Conclusions: Mice treated with DC loaded with apoptotic, syngeneic melanoma cells (DC/ApoB-16) or apoptotic, allogeneic melanoma cells (DC/ApoK-1735) had delayed tumor progression and improved survival compared to controls. CTL responses to B-16 cells were seen in both treatment groups, and there was no difference in tumor size, survival, or CTL responses among the treatment groups. We conclude that DC loaded with apoptotic allogeneic melanoma cells may be a practical and effective approach to polyvalent immunotherapy of melanoma. We are currently designing a clinical trial to evaluate the efficacy of this treatment in patients with high-risk disease.     

乳腺癌小鼠动物模型中白细胞介素-17的表达及其意义

目的 探讨小鼠乳腺癌动物模型中白细胞介素-17(IL-17)的表达及意义.方法 以MA782/5S28102及4T1细胞株建立两种乳腺癌动物模型.分别于接种后1周和4周采用Western blot检测肿瘤组织中IL-17的表达.使用PMA+CD3单抗+CD28单抗刺激后,采用酶联免疫吸附试验(ELISA)检测肿瘤细胞和淋巴细胞培养体系上清中IL-17的含量.同时观察IL-17对培养体系及4T1荷瘤小鼠中肿瘤细胞生长的影响.结果 乳腺癌模型中晚期肿瘤组织中IL-17的表达水平均明显较早期有所升高.分离的肿瘤细胞接受刺激后,几乎不分泌IL-17,淋巴细胞分泌大量IL-17(P＜0.01).IL-17不能促进4T1细胞的生长(增长率分别为1.11±0.11和1.28 ±0.21,P＞0.05);将IL-17输注至4T1荷瘤小鼠的体内,可见肿瘤生长速度明显加快(P＜0.05).输注IL-17的荷瘤小鼠肿瘤组织中微血管密度(MVD)明显增加(MVD分别为:35.79±9.49,13.52±3.55,P＜0.01).结论 IL-17在晚期肿瘤组织中明显升高,IL-17可能通过促进肿瘤组织内微血管形成加快肿瘤的生长.

Abstract：

Objective To explore the impact of interleukin (IL) -17 expression on tumor growth in experimental models of murine mammary carcinoma and potential mechanisms. Methods Two murine cell lines, MA782/5S28102 and 4T1 were used to establish experimental models of murine mammary carcinoma. The IL-17 expression in tumor tissues derived from MA782-bearing mice or 4T1-bearing mice was detected in early and late stages of the tumor by Western blotting. The tumor cells and tumor-infiltrated-lymphocytes were separated from tumor tissues and cultured for 5 days with stimulation of PMA, anti-CD3 antibody and anti-CD28 antibody. The supernatants of culture media of stimulated tumor cells or tumor-infiltrated-lymphocytes were harvested and tested for IL-17 concentration by enzyme linked immunosorbent assay (ELISA). To evaluate the effect of IL-17 on the proliferation of tumor cells, 4T1 cells were culture in media with or without IL-17 and the cell number was counted on the day 5. For ire vivo assay, 4T1-bearing mice were injected with IL-17 or culture media via tail vein, and the tumor volume was measured. To assay the angiogenesis, the tumor tissues from 4T1-bearing mice with or without injection of IL-17 were stained with anti-CD31 antibody by immunohistochemistry. Results The IL-17 expression was significantly higher in late stage than in early stage of tumor in two experimental models. The tumor expression of IL-17 was secreted by tumor infiltrated lymphocytes (P ＜0.01). IL-17 could not increase the generation of tumor cells in vitro (1. 11 ±0. 11, 1. 28 ±0. 21 ,P ＞0. 05). But IL-17, injected into 4T1 -bearing mice, markedly enhanced in vivo tumor growth and significantly increased tumor vascularity (35. 79 ±9. 49, 13. 52 ±3. 55,P ＜0.01). Conclusion IL-17 in tumor tissue probably promotes tumor growth through enhancing angiogenesis.     

Induction of PSA-specific CTLs and anti-tumor immunity by a genetic prostate cancer vaccine

BACKGROUND: Prostate cancer is the most common malignancy in Swedish and American men. Effective curative treatment modalities are debilitating and available only for localized disease. As an immunotherapy approach, DNA encoding prostate-specific antigen (PSA), was used to immunize mice and induce PSA-specific cellular immunity. METHODS: A plasmid expressing PSA, alone or in combination with plasmids coding for GM-CSF and/or IL-2, was used for DNA immunization. Cr-release, intracellular IFN-gamma cytokine staining, and tumor challenge assays were used to evaluate the immune response. RESULTS: The DNA vaccine induces PSA-specific cytotoxic T lymphocytes (CTLs) and when co-injected with IL-2 and GM-CSF it protects four of five mice against a PSA-expressing tumor challenge. CONCLUSIONS: We demonstrate that immunization with a PSA DNA vaccine can evoke PSA-specific cellular immune responses. We also show, for the first time, that a PSA DNA vaccine can induce anti-tumor immunity in vivo.