Enhancing the treatment effects of tumor cell purified autogenous heat shock protein 70-peptide complexes on HER-3-overexpressing breast cancer

Objective The aim of this study was to enhance the treatment effect of tumor purified autogenous heat shock protein 70-peptide complexes (HSP70-PCs) on HER-3-overexpressing breast cancer. Methods In this study, we first studied the expression of HER-3 in breast cancer tissues and its relationship with patient characteristics. We then purified HSP70-PCs from primary breast cancer cells with different HER-2 and HER-3 expression profiles and determined the cytotoxicity of autogenous dendritic cells (DCs) and CD8+ T cells induced by these complexes. Third, recombinant human HSP70-HER-3 protein complexes were used to inhibit the autogenous HSP70-PCs purified from HER-3–overexpressing breast cancer cells, and the resulting immunological response was examined. Results The results show that HSP70-PCs can be combined with recombinant HSP70-HER-3 protein complexes to induce stronger immunological responses than autogenous HSP70-PCs alone and that these treatments induce autogenous CD8+ T cell killing of HER-3-positive breast cancer cells. Conclusion These findings provide a new direction for HSP70-DC-based immunotherapy for patients with HER-3-overexpressing breast cancer.     

来源于人膀胱癌细胞株EJ的HSP70负载的DC所激发的特异性CTL细胞的体外应答效应

Objective: To investigate whether human dendritic cells (DC) derived from peripheral blood mononuclear cells (PBMC), which were pulsed by heat shock protein 70 (HSP70) isolated from human bladder tumor cell lines of EJ, were able to induce peptide specific cytotoxic T-lymphocytes (CTL) response in vitro and give the experimental foundation for the future clinical trials of immunotherapy in bladder tumor. Methods: The EJ-derived HSP70 co-cultured with DC from the healthy volunteers' PBMC, along with the crude lysate (the supernatant before HSP70 purification) from EJ cells were used as the experimental groups and DC not pulsed by any tumor cells antigen were the blank control. The autologous T-lymphocytes were added into the above various DC groups, and after incubation, the stimulation indexes (SI) and interferon-γ (IFN-γ) were detected to evaluate the immune activities of various DC groups. The killing effects of CTL to target cells, EJ and Hela cells, were determined with 51Cr releasing test. Results: Both DC/HSP70 and DC/the crude lysate could effectively activate CTL in vitro and kill target cells EJ. The killing effect of DC/HSP70 to EJ was much stronger than DC/the crude lysate (the supernatant before HSP70 purification) (P < 0.05). DC without any tumor cell antigens had a lower killing power to EJ. Meanwhile, DC/ HSP70 had little killing power to Hela non-relevant to bladder tumor histopathologically as compared with EJ cells (P < 0.05). Conclusion: The DC pulsed by HSP70 derived from the autologous tumor cells could induce a peptide complexes specific CTL response to tumor cells, and the CTL response induced by the DC/HSP70 was stronger, which display the basis of the possible clinical application of DC/HSP70 for bladder tumor.     

Heat shock protein 70: role in antigen presentation and immune stimulation

Heat shock proteins (HSP) when released into the extracellular milieu can act simultaneously as a source of antigen due to their ability to chaperone peptides and as a maturation signal for dendritic cells, thereby inducing DCs to cross-present antigens to CD8+ T-cells. HSP can also act independently from associated peptides, stimulating the innate immune system. Previous results regarding the activation of NK cells by HSP70 cell surface expression on tumour cells and soluble HSP70 will be further covered elsewhere within this issue. For cross-presentation, HSP70-peptide complexes (HSP70-PC) were used from two human melanoma cell lines that differ in the expression of the tumour-associated antigen tyrosinase. Purified HSP70-PC consists of both the constitutively expressed HSC70 and the inducible HSP70. HSP70-peptide complexes purified from tyrosinase positive (HSP70-PC/tyr + ) human melanoma cells, incubated with immature DCs, results in the activation of HLA-*A0201-restricted tyrosinase peptide-specific T-cells. Receptor-mediated uptake of HSP70-PC by DCs and intracellular transport are required for efficient MHC class I restricted cross-presentation of chaperoned peptides. Demonstration of HSP70-PC mediated cross-presentation of such non-mutated naturally expressed tumour antigens is of special clinical interest with regard to hyperthermia. Tumour regression and improved local control have been shown within clinical phase II/III trials integrating regional hyperthermia combined with radiation and/or chemotherapy in multimodal treatment strategies. According to the proposed concept, local necrosis induced by hyperthermic treatment induces the release of HSPs, followed by uptake, processing and presentation of associated peptides by DCs. By acting as chaperone and a signal for DC maturation, HSP70-PC might efficiently prime circulating T-cells. Therefore, upregulating HSP70 and causing local necrosis in tumour tissue by hyperthermia offers great potential as a new approach to directly activate the immune system.     

Heat shock protein 70: role in antigen presentation and immune stimulation.

Heat shock proteins (HSP) when released into the extracellular milieu can act simultaneously as a source of antigen due to their ability to chaperone peptides and as a maturation signal for dendritic cells, thereby inducing DCs to cross-present antigens to CD8+ T-cells. HSP can also act independently from associated peptides, stimulating the innate immune system. Previous results regarding the activation of NK cells by HSP70 cell surface expression on tumour cells and soluble HSP70 will be further covered elsewhere within this issue. For cross-presentation, HSP70-peptide complexes (HSP70-PC) were used from two human melanoma cell lines that differ in the expression of the tumour-associated antigen tyrosinase. Purified HSP70-PC consists of both the constitutively expressed HSC70 and the inducible HSP70. HSP70-peptide complexes purified from tyrosinase positive (HSP70-PC/tyr+) human melanoma cells, incubated with immature DCs, results in the activation of HLA-*A0201-restricted tyrosinase peptide-specific T-cells. Receptor-mediated uptake of HSP70-PC by DCs and intracellular transport are required for efficient MHC class I restricted cross-presentation of chaperoned peptides. Demonstration of HSP70-PC mediated cross-presentation of such non-mutated naturally expressed tumour antigens is of special clinical interest with regard to hyperthermia. Tumour regression and improved local control have been shown within clinical phase II/III trials integrating regional hyperthermia combined with radiation and/or chemotherapy in multimodal treatment strategies. According to the proposed concept, local necrosis induced by hyperthermic treatment induces the release of HSPs, followed by uptake, processing and presentation of associated peptides by DCs. By acting as chaperone and a signal for DC maturation, HSP70-PC might efficiently prime circulating T-cells. Therefore, upregulating HSP70 and causing local necrosis in tumour tissue by hyperthermia offers great potential as a new approach to directly activate the immune system.     

Paucity of functional T-cell memory to melanoma antigens in healthy donors and melanoma patients.

The functional characteristics of CD8+ T cells specific for melanoma antigens (MAs) have often been defined after in vitro culture using nonprofessional antigen-presenting cells. We have examined CD8+ T-cell immunity to MAs and a viral antigen (influenza) in uncultured T cells of healthy donors and melanoma patients using autologous, mature, monocyte-derived dendritic cells (DCs) pulsed with peptide antigens and viral vectors. Antigen-specific IFN-gamma-producing T cells reactive with HLA-A*0201-restricted peptides from four melanoma antigens (MelanA/MART-1, MAGE-3, tyrosinase, and gp100) were detected only at low frequencies (<30 per 2 x 10(5) peripheral blood mononuclear cells for each of the MAs) from HLA-A2.1-positive healthy donors (n = 12) and patients with stages III/IV melanoma (n = 8). Detection of MA-specific, but not influenza matrix peptide (Flu-MP)-specific, T cells required a high concentration (10 microg/ml) of the peptide in this assay. Furthermore, these T cells did not recognize endogenously processed antigen on tumor cell lines or cells infected with viral vectors capable of expressing MAs. The use of autologous, mature DCs led to a significant increase in the number of Flu-MP, but not MA-specific, T cells in 16-h ELISPOT assays for both melanoma patients and healthy donors. In 1-week cocultures with DCs pulsed with 10 microg/ml peptide, MelanA/MART-1-specific T cells did not readily proliferate or differentiate into lytic effectors, in contrast to strong influenza-specific lytic responses. Therefore, despite distinct memory responses to influenza antigens, melanoma patients and healthy controls have a paucity of MA-reactive memory T cells, failing to rapidly generate IFN-gamma-secreting lytic effectors in short-term assays, even when stimulated by DCs.     

Human tumor cells killed by anthracyclines induce a tumor-specific immune response

Immunogenic cell death is characterized by the early surface exposure of chaperones including calreticulin and HSPs, which affect dendritic cell (DC) maturation and the uptake and presentation of tumor antigens. It has also been shown that it is characterized by the late release of high mobility group box 1 (HMGB1), which acts through Toll-like receptor 4 (TLR4) and augments the presentation of antigens from dying tumor cells to DCs. Most of the data on immunogenic tumor cell death were obtained using mouse models. In this study, we investigated the capacity of clinically used chemotherapeutics to induce immunogenic cell death in human tumor cell lines and primary tumor cells. We found that only anthracyclines induced a rapid translocation of calreticulin, HSP70, and HSP90 to the cell surface and the release of HMGB1 12 hours after the treatment. The interaction of immature DCs with immunogenic tumor cells led to an increased tumor cell uptake and induces moderate phenotypic maturation of DCs. Killed tumor cell-loaded DCs efficiently stimulated tumor-specific IFN-γ-producing T cells. DCs pulsed with killed immunogenic tumor cells also induced significantly lower numbers of regulatory T cells than those pulsed with nonimmunogenic tumor cells. These data indicate that human prostate cancer, ovarian cancer, and acute lymphoblastic leukemia cells share the key features of immunogenic cell death with mice tumor cells. These data also identify anthracyclines as anticancer drugs capable of inducing immunogenic cell death in sensitive human tumor cells.     

High hydrostatic pressure induces immunogenic cell death in human tumor cells

Recent studies have identified molecular events characteristic of immunogenic cell death (ICD), including surface exposure of calreticulin (CRT), the heat shock proteins HSP70 and HSP90, the release of high‐mobility group box protein 1 (HMGB1) and the release of ATP from dying cells. We investigated the potential of high hydrostatic pressure (HHP) to induce ICD in human tumor cells. HHP induced the rapid expression of HSP70, HSP90 and CRT on the cell surface. HHP also induced the release of HMGB1 and ATP. The interaction of dendritic cells (DCs) with HHP‐treated tumor cells led to a more rapid rate of DC phagocytosis, upregulation of CD83, CD86 and HLA‐DR and the release of interleukin IL‐6, IL‐12p70 and TNF‐α. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor‐specific T cells. DCs pulsed with HHP‐treated tumor cells also induced the lowest number of regulatory T cells. In addition, we found that the key features of the endoplasmic reticulum stress‐mediated apoptotic pathway, such as reactive oxygen species production, phosphorylation of the translation initiation factor eIF2α and activation of caspase‐8, were activated by HHP treatment. Therefore, HHP acts as a reliable and potent inducer of ICD in human tumor cells.     

Heat-Shock Protein 70 as a Tumor Antigen for in vitro Dendritic Cell Pulsing in Renal Cell Carcinoma Cases

Immunological functions of heat shock proteins (HSPs) have long been recognized. In this study we aimed toefficiently purify HSP70 from renal cell carcinoma and test it as a tumor antigen for pulsing dendritic cells in vitro.HSP70 was purified from renal cell carcinoma specimens by serial column chromatography on Con A-sepharose,PD-10, ADP-agarose and DEAE-cellulose, and finally subjected to fast protein liquid chromatography (FPLC).Dendritic cells derived from the adherent fraction of peripheral blood mononuclear cells were cultured in thepresence of IL-4 and GM-CSF and exposed to tumor HSP70. After 24 hours, dendritic cells were phenotypicallycharacterized by flow cytometry. T cells obtained from the non-adherent fraction of peripheral blood mononuclearcells were then co-cultured with HSP70-pulsed dendritic cells and after 3 days T cell cytotoxicity towards primarycultured renal cell carcinoma cells was examined by Cell Counting Kit-8 assay. Dendritic cells pulsed in vitrowith tumor-derived HSP70 expressed higher levels of CD83, CD80, CD86 and HLA-DR maturation markersthan those pulsed with tumor cell lysate and comparable to that of dendritic cells pulsed with tumor cell lysateplus TNF-α. Concomitantly, cytotoxic T-lymphocytes induced by HSP70-pulsed dendritic cells presented thehighest cytotoxic activity. There were no significant differences when using homologous or autologous HSP70as the tumor antigen. HSP70 can be efficiently purified by chromatography and induces in vitro dendritic cellmaturation in the absence of TNF-α. Conspecific HSP70 may effectively be used as a tumor antigen to pulsedendritic cells in vitro.     

Demonstration of a well-characterized tumor-associated antigen on melanoma cell surface

We have isolated and characterized a melanoma tumor-associated antigen (TAA) from the spent culture medium of a melanoma cell line. Its presence has been detected in 72% of different melanoma specimens but not in normal tissues. Because tumor antigens may stimulate or perhaps block immune reactions to cancer cells, their presence on the cell surface may be a critical factor influencing tumor growth. Therefore, to define further the immunobiological characteristics of melanoma TAA, this antigen was investigated by membrane immunofluorescence. Serum from a melanoma patient known to have a high anti-TAA antibody titer was absorbed quantitatively with lymphoblastoid cells autologous to the target melanoma cells to remove non-anti-TAA antibodies. The absorbed serum was reacted with three cultured melanoma cell lines, UCLA-SO-10 (M10), UCLA-SO-14(M), and UCLA-SO-24 (M24). Of these cell lines, M10 and M14 are known to express the antigen as assessed by radioimmunoassay. Reaction of the antibody(s) to the antigen(s) on the melanoma cell surface was detected by fluorescein-conjugated goat anti-human IgG. The reactivity of the absorbed serum was inhibited by preincubation with purified melanoma TAA. These results clearly demonstrate that melanoma TAA is expressed on the cell surface of cultured melanoma cells.     

Hyperthermia improves cellular immune response to human hepatocellular carcinoma subsequent to co-culture with tumor lysate pulsed dendritic cells

Dendritic cells play a major role in cellular immunity. The crucial steps of antigen presentation and processing by DCs may be limiting factors for adoptive cellular immunotherapy. Here, we investigated whether hyperthermia of human hepatocellular carcinoma (HCC) cells induces enhanced cytotoxic cellular immune response. Peripheral blood mononuclear cell (PBMC)-derived DCs were pulsed with tumor cell lysate of the human HCC cell line HepG2, which had been heat shocked prior to incubation for 5 h. Subsequent to TNFalpha-induced maturation DCs were co-cultured with autologous CD4+ and/or CD8+ cells, and T cell mediated cytolysis of HepG2 cells was assessed. We observed enhanced CD4+/8+ cellular cytotoxicity against HepG2 cells subsequent to co-culture with the heat shocked tumor lysate pulsed DCs as compared to pulsing DCs with lysate of non-heat shocked tumor cells. The improved cellular immune response can be related to enhanced expression of HSP 70 and 90 in HepG2 cells upon hyperthermia.     

Human autologous tumor-specific T cells in malignant melanoma

T cell lines and clones with autologous tumor-specific activity have been developed in malignant melanoma by stimulating peripheral blood lymphocytes (PBL), lymph node lymphocytes or tumor-infiltrating lymphocytes (TIL) with autologous melanoma cells in the presence of recombinant interleukin 2 (rIL2). T-cell lines and clones have been developed with specific cytotoxicity and/or proliferative responses for autologous melanoma targets but not for allogeneic melanoma tumor cells, autologous normal cells or natural killer (NK)-sensitive targets. The concentration of rIL2 is critical for the generation of autologous tumor-specific T-cell lines, with low rIL2 concentrations (up to 800 IU/ml) facilitating the growth of T-cell lines with tumor-specific activity. The T-cell receptor (TCR) and the CD3 antigen are involved in specific cytotoxicity and/or proliferative responses of these T-cell lines and clones. An oligoclonal pattern of -chain TCR gene rearrangements was observed on T-cell lines and clones with autologous tumor-specific cytotoxicity, suggesting that they are comprised of T cells that have undergone a clonal expansion in response to particular antigen. Autologous tumor-specific cytotoxic T cells are HLA-restricted and recognize on the melanoma tumor cells HLA Class I or possibly Class II antigens plus a tumor-specific determinant. TIL from patients with metastatic melanoma have unique characteristics in comparison with PBL and lymph node lymphocytes and they appear to contain substantial proportions of T cells that have been locally sensitized to autologous tumor cells. Single stimulation of TIL with autologous tumor cells in the presence of rIL2 is sufficient for the generation of T cell lines with autologous tumor-specific activity, whereas, multiple stimulation of PBL and lymph node lymphocytes was required to achieve the same purpose. TIL-derived T cell lines have been expanded in rIL2 in vitro by at least 1,500-fold without losing their activity. Approximately, 40% of the patients exhibited complete or partial responses to adoptive immunotherapy with melanoma TIL and rIL2.     

A CD4+, HLA-DR7-restricted T-helper lymphocyte clone recognizes an antigen shared by human malignant melanoma and glioma

CD4(+) Th cells that are restricted by MHC class II molecules play an important role in the induction of antitumor immune responses. We have established a stable CD4(+) Th cell clone (Th35-1A) from the PBMCs of a patient with primary cutaneous melanoma. The Th cell clone is noncytolytic and proliferates specifically in the presence of irradiated autologous melanoma cells or autologous EBV-transformed B cells pulsed with melanoma tumor cell lysates. Th35-1A produces IFN-gamma (a Th1-type cytokine) after autologous tumor cell stimulation, and its proliferative reactivity is HLA class II-restricted. Th cells showed helper activity for PWM responses of PBMCs. Using a panel of HLA class II-matched and unmatched EBV-B cells as APCs and allogeneic melanoma tumor cell lysate as stimulant, DR7 was delineated as the HLA class II restriction element used by the Th cell clone. In agreement with these results, transfection of an allogeneic melanoma cell line with HLA-DR7 isolated from autologous EBV-B cells rendered the cell line stimulatory for Th35-1A cells. Specificity studies using autologous EBV-B cells (EBV-B35) pulsed with a panel of allogeneic tumor cell lysates of various tissue origins indicated that the Th cell clone recognizes an antigen shared by melanoma and glioma cells. The availability of the Th cell clone may lead to the development of new therapies against melanoma, using adoptive Th cell transfer and/or active immunization with a shared Th cell antigen.     

Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine

Immunization to multiple defined tumor antigens for specific immune therapy of human cancer has thus far proven difficult. Eighteen HLA A*0201(+) patients with metastatic melanoma received injections s.c. of CD34(+)progenitor-derived autologous dendritic cells (DCs), which included Langerhans cells. DCs were pulsed with peptides derived from four melanoma antigens [(MelAgs) MelanA/MART-1, tyrosinase, MAGE-3, and gp100], as well as influenza matrix peptide (Flu-MP) and keyhole limpet hemocyanin (KLH) as control antigens. Overall immunological effects were assessed by comparing response profiles using marginal likelihood scores. DC injections were well tolerated except for progressive vitiligo in two patients. DCs induced an immune response to control antigens (KLH, Flu-MP) in 16 of 18 patients. An enhanced immune response to one or more MelAgs was seen in these same 16 patients, including 10 patients who responded to >2 MelAgs. The two patients failing to respond to both control and tumor antigens experienced rapid tumor progression. Of 17 patients with evaluable disease, 6 of 7 patients with immunity to two or less MelAgs had progressive disease 10 weeks after study entry, in contrast to tumor progression in only 1 of 10 patients with immunity to >2 MelAgs. Regression of >1 tumor metastases were observed in seven of these patients. The overall immunity to MelAgs after DC vaccination is associated with clinical outcome (P = 0.015).     

Gangliosides of human melanoma: altered expression in vivo and in vitro

The ganglioside composition of human melanoma was analyzed in five sets of tumor specimens obtained directly from surgery, from the autologous tissue culture cell lines, and from the autologous cell lines grown in athymic nude mice. Total gangliosides of these 15 melanoma specimens were isolated and purified, and the amount of each component ganglioside was analyzed by thin-layer chromatography and a thin-layer chromatography scanner. The ganglioside composition of the five surgical melanoma specimens clearly exhibited different patterns from each other. Moreover, none of the autologous cultured melanomas possessed the same ganglioside composition as their original biopsied tumors. However, when these melanoma cell lines were transplanted into nude mice, the ganglioside composition was converted back to the same ganglioside pattern as in the original surgical specimens. The results support the view that changes in the ganglioside composition of melanoma during in vitro growth are caused by the culture environment rather than by selection of melanoma cells with a particular genotype. Reestablishment of the original ganglioside patterns after passage in nude mice provides clear evidence that in vivo expression of gangliosides is a conserved and stable function specified by the human melanoma cells.     

HIFU治疗后肿瘤抗原对树突状细胞的活化及其抗肿瘤效应

目的：探讨HIFU治疗小鼠H22抑制性肝癌后产生的肿瘤抗原对机体抗肿瘤免疫功能增强的机制。方法：正常小鼠骨髓中提取骨髓细胞,在rmIL-4、rmGM-CSF奈件下培养7d,制备小鼠骨髓树突状细胞,用HIFU治疗小鼠移植性肝癌后产生的肿瘤抗原活化树突状细胞,再用活化后的树突状细胞激活T淋巴细胞为细胞毒性T细胞,用MTT法检测CTL在体外特异性杀伤肿瘤靶细胞的能力。结果：B16肿瘤HSP70-肽复合物组和H22肿瘤HSP70-肽复合物组的脾淋巴细胞的增殖率均高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组,P〈0．001;但两组之间差异无统计学意义,P〉0．05。H22肿瘤HSP70-肽复合物组CTL对H22肿瘤细胞的杀伤率为70．0％,明显高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组（P〈0．001）,但对非靶细胞B16肿瘤的杀伤率与上述各组的差异无统计学意义;B16肿瘤HSP70-肽复合物组CTL对B16肿瘤细胞的杀伤率为78．5％,对H22细胞的杀伤率为21．4％,表明CTL对肿瘤细胞的杀伤作用具有特异性。结论：HIFU治疗后坏死肿瘤组织中的HSP70-肽复合物作为肿瘤疫苗,通过活化DC和刺激T淋巴细胞增殖为CTL,发挥特异性抗肿瘤免疫功能。     

体外构建的HTA-HSP70BCG冲激的树突状细胞疫苗的抗肿瘤作用

 目的 观察体外构建的榄香烯复合瘤苗抗原-卡介苗热休克蛋白70复合物（H_TA—HSP70_BCG）诱导的树突状细胞疫苗的抗肿瘤效应。方法 来源于小鼠的肝癌Hca-F榄香烯复合疫苗的抗原（HTA）与卡介苗来源的HSPT0（HSP70_BCG）在体外构建成HTA—HSP70_BCG复合物，用GM-CSF和IL-4诱导树突状细胞（DCs），分别用HTA—HSP70_BCG、HTA和HSP70麟对其冲激。用舯法检测该DCs刺激的全脾细胞的增殖活性及被刺激的脾细胞的细胞毒活性。用流式细胞仪检测DCs表面（瑚6和CD40的表达。结果 体外构建HTA—HSP70_BCG可以诱导DCs成熟，表现为DCs表达CD86和CD40上调，该DCs可以刺激全脾细胞增殖并使其产生特异性杀瘤活性，其强度明显大于HTA。结论 体外构建HTA—HSP70麟复合物可以诱导DCs成熟，该DCs可以激活脾细胞产生较强的特异性抗瘤效应。     

Vaccination with melanoma lysate-pulsed dendritic cells, of patients with advanced colorectal carcinoma: report from a phase I study

Immune therapy have shown new and exciting perspectives for cancer treatment. Aim of our study was to evaluate toxicity and possible adverse effects from vaccination of patients with advanced colorectal cancer with autologous dendritic cells (DC) pulsed with lysate from a newly developed melanoma cell line, DDM-1.13. Six patients were enrolled in the phase I trial. Autologous DCs were generated in vitro from peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were pulsed with melanoma cell lysate from a cloned and selected melanoma cell line enriched in expression of MAGE-A antigens and deficient in expression of melanoma differentiation antigens: tyrosinase, MART-1 and gp100. Vaccinations were administered intradermally on the proximal thigh with a total of five given vaccines at 2 weeks intervals. Each vaccine contained 3-5 x 10(6) DCs. Five of the six patients received all five vaccines. The treatment was well tolerated in all patients without any observed vaccine-correlated adverse effects. Treatment with this DC-based cancer vaccine proved safe and non-toxic.     

Identification of C-met oncogene as a broadly expressed tumor-associated antigen recognized by cytotoxic T-lymphocytes.

PURPOSE: C-Met proto-oncogene is a receptor tyrosine kinase that mediates the oncogenic activities of the hepatocyte growth factor. Using a DNA chip analysis of tumor samples from patients with renal cell carcinoma and sequencing of peptides bound to the HLA-A*0201 molecules on tumor cells a peptide derived from the c-Met protein was identified recently. EXPERIMENTAL DESIGN: We used this novel HLA-A*0201 peptide for the induction of specific CTLs to analyze the presentation of this epitope by malignant cells. RESULTS: The induced CTL efficiently lysed target cells pulsed with the cognate peptide, as well as HLA-A*0201-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Furthermore, the induced c-Met-specific CTLs recognized autologous dendritic cells (DCs) pulsed with the peptide or transfected with whole-tumor mRNA purified from c-Met-expressing cell lines. We next induced c-Met-specific CTLs using peripheral blood mononuclear cells and DC from an HLA-A*0201-positive patient with plasma cell leukemia to determine the recognition of primary autologous malignant cells. These CTLs lysed malignant plasma cells while sparing nonmalignant B- and T-lymphocytes, monocytes, and DCs. CONCLUSION: Our results demonstrate that c-Met oncogene is a novel tumor rejection antigen recognized by CTL and expressed on a broad variety of epithelial and hematopoietic malignant cells.     

Dead or alive: immunogenicity of human melanoma cells when presented by dendritic cells

Uveal melanoma is an aggressive malignancy with a poor prognosis despite current therapeutic intervention. These tumors have been shown to be antigenic because they express a number of melanoma-associated antigens and are therefore attractive targets for immunotherapy. Here, we investigated the immunogenicity of uveal melanoma cells that have undergone apoptosis and compared this with their necrotic or live counter-parts. The fate of the tumor antigens in these cells largely depends on their ability to be processed and phagocytosed by dendritic cells (DCs). Flow cytometric analysis shows that human DCs form conjugates more efficiently with dead uveal melanoma cells, and consequently these are effective stimuli of lymphocyte proliferation. However, only DCs pulsed with apoptotic cells were able to induce proliferation of CD8+ cytotoxic T cells and stimulate antigen-specific T cells. This study demonstrates for the first time that DCs derived from melanoma patients process and present antigens derived from both HLA-matched or HLA-mismatched human apoptotic tumor cells stimulating both CD4+ and CD8+ T cells. This approach may be important to the development of DC-based immunotherapies for melanoma.