Modification of the HER2/NEU-derived tumor antigen GP2 improves induction of GP2-reactive cytotoxic T lymphocytes.

GP2 (IISAVVGIL), the p654-662 HER2/neu-derived tumor antigen, induces HLA-A2-restricted cytotoxic T lymphocytes (CTL) reactive to various epithelial cancers. The binding affinity of GP2 for HLA-A2, however, is very low. To improve the immunogenicity of GP2, we tested 10 different amino acid substitutions into GP2 at the C- and N- terminus. Five out of 10 modified peptides, especially those containing phenylalanine at position 1 (1F), showed a significantly improved binding affinity to HLA-A2. 1F-based modified peptides were well recognized by GP2-specific CTL. These peptides were used to stimulate peripheral blood lymphocytes from HLA-A2 healthy donors using peptide-pulsed autologous dendritic cells (DC). After 3 or more weekly stimulations, CTL activity against GP2 pulsed T2 (T2-GP2) and HER2/neu-overexpressing tumor cells was measured in (51)Cr release and IFN-gamma secretion assays. The modified peptides significantly enhanced GP2-specific CTL activity in some donors. In particular, the peptide with phenylalanine at position 1, leucine at position 2 and valine at position 10 (1F2L10V) maximized the CTL activity against both T2-GP2 and HER2/neu-positive tumor cells. Peptide 1F2L10V increased not only the binding affinity to HLA-A2 but also improved recognition of GP2. These data suggest that DC + modified GP2 may improve immune therapies for the treatment of HER2/neu overexpressing tumors.

Loss of T-cell receptor-CD3zeta and T-cell function in tumor-infiltrating lymphocytes but not in tumor-associated lymphocytes in ovarian carcinoma.

BACKGROUND: Impaired T-cell function has been noted in tumor-infiltrating lymphocytes (TIL). Recently, loss of function was found to be associated with modifications in T-cell receptor complex (TCR)-mediated signaling. A common feature is loss or reduced expression levels of the signaling chain, TCRzeta. We evaluated whether loss of function in TIL and tumor-associated lymphocytes (TAL) from patients with ovarian cancer is associated with changes in TCRzeta expression, and which factors can cause these defects. METHODS: TIL and TAL were isolated from multiple patients and evaluated for their proliferative capacity by stimulation with a polyclonal stimulus. In addition, expression of TCRzeta and CD3epsilon was evaluated in fresh TIL and TAL by the Western blot technique. Finally, various conditions within a tumor environment were tested for their effect on TCRzeta and CD3epsilon. RESULTS: TIL, but not TAL, were significantly impaired in their proliferative response, even when both populations were derived from the same patient (P <.05). Reduced proliferation levels were associated with loss of expression of TCRzeta but not of CD3epsilon. Exposure of normal T cells to relative ischemia or heat shock, or culture in medium without IL-2, did not significantly reduce expression of TCRzeta compared with CD3epsilon. However, coculture of T cells with tumor-derived macrophages or tumor-derived factors led to a selective loss of TCRzeta compared with CD3epsilon (P <.05). Further analysis suggested that oxides such as hydrogen peroxide secreted by macrophages may be responsible for loss of TCRzeta and high molecular weight factors secreted by certain tumors. CONCLUSIONS: TIL but not TAL show impaired T-cell function, which is associated with loss of TCRzeta. In addition to macrophages secreting oxides, loss of TCRzeta may be caused by tumor-derived soluble factors.

Expression and function of galectin-3, a beta-galactoside-binding protein in activated T lymphocytes.

A soluble beta-galactoside-binding lectin, galectin-3 has been shown to be involved in cell adhesion and activation of immune cells. Although galectin-3 is known to be expressed in various types of cells, it has not been shown whether galectin-3 is expressed in T lymphocytes. We present evidence here that galectin-3 is expressed in activated murine T lymphocytes including CD4+ and CD8+ T cells but not in resting T cells. Galectin-3 expression was induced by anti-CD3 mAb or mitogen and enhanced by common gamma-chain signaling cytokines, IL-2, IL-4, and IL-7, in activated T lymphocytes, whereas the inflammatory cytokines including TNF-alpha and IFN-gamma did not. Galectin-3 expression and proliferation were down-regulated by withdrawal of IL-2 and gamma irradiation. Antisense but not sense phosphorothioated oligonucleotides for galectin-3 inhibited galectin-3 expression and blocked proliferation of T cells significantly. This study suggests that up-regulation of galectin-3 plays an important role in proliferation of activated T lymphocytes.

B7.1 costimulation increases T-cell proliferation and cytotoxicity via selective expansion of specific variable alpha and beta genes of the T-cell receptor.

BACKGROUND: Optimal T-cell activation requires not only ligation of the T-cell receptor (TcR) but also delivery of costimulatory signals by various accessory molecules. The interaction of the costimulatory molecule B7.1 (CD80) with its receptor CD28 provides a strong positive signal to T cells. METHODS: The B7.1 gene was transduced into cultured human ovarian, breast, and pancreatic tumor cells by using a retroviral vector. Autologous as well as allogeneic naive T-cells were stimulated with either wild-type or B7.1-transduced tumor cells in a mixed lymphocyte tumor cell culture (MLTC). In addition to cytolytic activity, T-cell proliferation, T-cell subset composition, and the frequencies of TcR variable (V) alpha and beta genes were compared in T cells from both types of MLTC. RESULTS: Introduction of the B7.1 gene into tumor cells was successful in all tumors to a varying degree. Those tumors expressing high levels of B7.1 induced significantly higher levels of T-cell proliferation than wild-type tumor cells. T-cell subset composition did not markedly differ between T cells stimulated with wild-type tumor cells or B7.1-expressing tumor cells. However, T cells stimulated with B7.1-expressing tumor cells showed a significantly increased cytolytic potential. The increased cytotoxic T lymphocyte activity was associated with a higher frequency of specific TcR V alpha and V beta genes. In addition, B7.1 costimulation promoted oligoclonality among the responding T cells. CONCLUSIONS: These data suggest that costimulation through B7.1 promotes T-cell proliferation and cytotoxic activity through clonal expansions of T cells bearing antigen-specific TcR V alpha and V beta genes and through promotion of oligoclonality. The data also suggest that promoting B7.1-mediated costimulation is an important aspect of immune therapies.

Pancreatic cancer associated ascites-derived CTL recognize a nine-amino-acid peptide GP2 derived from HER2/neu.

BACKGROUND: The proto-oncogene HER2/neu encodes a 185 kDa transmembrane protein with extensive homology to the epidermal growth factor receptor. It is overexpressed in several human cancers of epithelial origin, such as pancreatic cancer. Previously, we demonstrated that CTL derived from breast, ovarian, and non-small cell lung cancer recognized a peptide derived from HER2/neu. The aim of this study was to evaluate whether this HLA-A2-binding peptide is a TAA in pancreatic cancer and if pancreatic cancer associated T-lymphocytes (TAL) are useful to generate tumor- and peptide-specific CTL. MATERIALS AND METHODS: TAL from malignant ascites of a HLA-A2+ pancreatic cancer patient whose tumor overexpressed HER2/neu were stimulated on solid-phase anti-CD3 and cultured in low-dose IL-2. Using repetitive autologous tumor cell stimulation, CTL were generated. RESULTS: CTL recognized autologous and allogeneic HER2/neu+ tumor cells in an HLA-A2 restricted fashion significantly. Furthermore, all CTL recognized p654-662 (GP2) derived from HER2/neu, but not the control peptide. CONCLUSIONS: These results demonstrate that this HER2/neu derived peptide is a TAA in pancreatic carcinoma. The identification of the HER2/neu derived peptide GP2 as a TAA in pancreatic cancer provides an opportunity for the design of novel immunotherapy and vaccine strategies. The possibility of generating peptide-specific CTL from malignant ascites enables future studies to identify more antigens in this disease.

Soluble MUC1 secreted by human epithelial cancer cells mediates immune suppression by blocking T-cell activation.

Solid tumors may secrete factors that mediate immune suppression in patients. We investigated the effect of supernatants from 25 human tumor cell lines on T-lymphocytes from healthy donors. A profound inhibition of proliferation, cytokine secretion and cytotoxic activity was seen when T-cells were cultured in concentrated tumor supernatants from 6 cell lines fractionated into high (>100 kDa) m.w. molecules. Interestingly, the inhibitory effects were reversed when the tumor supernatant was removed. Cell cycle studies of inhibited T-cells showed most of them were growth arrested in the G(0)/G(1) phase similar to naïve T-cells. In addition, these T-cells did not express IL2-receptors and expression of CD54 (ICAM-1) and CD58 (LFA-3) resembled that of resting T-cells. Protein gel electrophoresis of the tumor supernatants and western blot analysis demonstrated the presence of soluble MUC1 in the inhibitory tumor supernatants but not in control supernatant. Most importantly, depletion of soluble MUC1 by immunoprecipitation from the tumor supernatants neutralized the inhibitory effects on T-lymphocytes. Therefore, our results show that MUC1 shed by cultured epithelial tumor cells mediates inhibition of T-cell proliferation and function by inducing cell growth arrest.

Local secretion of IFN-gamma induces an antitumor response: comparison between T cells plus IL-2 and IFN-gamma transfected tumor cells.

Previously we described that the adoptive transfer of tumor-infiltrating lymphocytes (TIL) + interleukin-2 (IL-2) leads to eradication of established methylcholanthrene (MCA)-105 fibrosarcoma pulmonary metastases in a congenic murine model. The in vivo efficacy of TIL was associated with their ability to secrete interferon-gamma (IFN-gamma), and to a lesser extent granulocyte-macrophage colony-stimulating factor. The local secretion of these cytokines resulted in recruitment of naive host immune cells to the tumor and eventually in a successful host antitumor immune response. In the present study, to further evaluate the role of IFN-gamma in the induction of a host antitumor immune response, we compared the treatment efficacy of adoptively transferred T cells and IFN-gamma gene transfected tumor cells (MCA-105/IFN-gamma) as delivery systems of IFN-gamma. Treatment with TIL-IL-2 or irradiated MCA-105/IFN-gamma induced a similar reduction in pulmonary metastases of MCA-105 tumor. In contrast, irradiated wild-type MCA-105 or TIL from IFN-gamma gene knockout mice did not cause tumor eradication. MCA-105 tumor-bearing mice treated with MCA-205/IFN-gamma showed a partial reduction in the number of pulmonary metastases. Histologically, lungs of successfully treated mice showed that initially activated macrophages expressing inducible nitric oxide synthase (iNOS) and dendritic cells infiltrated the tumor bed. Subsequently, CD4+ and CD8+ T cells infiltrated tumors. The therapeutic efficacy of IFN-gamma transfected tumor cells was eliminated when either CD4+ T cells or CD8+ T cells were depleted. These results suggest that local secretion of IFN-gamma induces a tumor-specific host antitumor immune response mediated through activated macrophages, dendritic cells, and tumor-specific T cells. This may be a common component of successful immunotherapy.

Tumor-specific cytokine release by donor T cells induces an effective host anti-tumor response through recruitment of host naive antigen presenting cells.

We recently reported that tumor eradication induced by immunotherapy (IT) in a congenic mouse model using tumor infiltrating lymphocytes (TIL) + recombinant interleukin-2 (rIL-2) is dependent on recruitment of naive host immune cells at the tumor sites. The recruitment of host immune cells was induced mainly through a local secretion of interferon-gamma (IFN-gamma) produced by donor T cells. We now further investigated how a non-specific inflammatory response progresses to a host T-cell-mediated tumor-specific response. In cross-over experiments using MCA-105 and MCA-205 sarcoma tumors, pulmonary metastatic disease was eradicated only in mice treated with tumor-matched TIL + rIL-2. In vitro, TIL stimulated with the tumor of origin secreted relatively high levels of IFN-gamma and granulocyte-macrophage colony stimulating factor (GM-CSF) compared to TIL stimulated with mismatched tumor cells. In lungs of tumor-bearing mice treated with matched TIL + rIL-2, significant increases in the percentages of IFN-gamma, GM-CSF and tumor necrosis factor-alpha (TNF-alpha) positive cells were detected, as well as of macrophages, natural killer (NK) cells and dendritic cells. Depletion of macrophages or NK cells did not inhibit the efficacy. In contrast, depletion of dendritic cells partially inhibited the efficacy of the treatment. Combined depletion of dendritic cells and macrophages abrogated more than 80% of the efficacy. Our data suggest that successful IT may require 3 steps: (1) release of inflammatory cytokines by donor TIL after restimulation by tumor cells; (2) infiltration of host immune cells in response to local cytokine production; and (3) activation of tumor-specific host immune cells by dendritic cells and to a lesser extent by macrophages.

T cells mediate treatment of six-day-old cytokine-gene-transfected mouse mammary tumor.

We have previously shown that immunologically different mouse mammary cancer cell lines induce antitumor responses after IL2 or IL4 gene transfection. We now report the ability of cytokine-transfected tumors to induce eradication of established wild-type tumor. Animals with 6-day-old tumor treated with IL2-transfected cells also had significantly smaller tumors 2.8 and 1.7 cm2 (EMT6 and 410.4). Findings were similar for IL4-transfected cells. Tumor infiltrating lymphocytes or cells from draining lymph nodes demonstrated tumor-specific in vitro cytotoxicity. Immunohistochemical studies revealed T cell infiltrates in transfected tumors.

Pancreatic cancer cells can evade immune surveillance via nonfunctional Fas (APO-1/CD95) receptors and aberrant expression of functional Fas ligand.

BACKGROUND: The Fas (APO-1/CD95) receptor/Fas ligand (FasR/FasL) system plays a key role in immune surveillance. We investigated the possibility of a tumor escape mechanism involving the FasR/FasL system in pancreatic cancer cells. METHODS: Fourteen pancreatic cancer cell lines and 3 pancreatic cancer surgical specimens were studied for their expression of FasR and FasL by flow cytometry, immunoblotting, and immunohistochemistry, FasR function was tested with an anti-FasR antibody. FasL function was assessed by coculture assays using pancreatic cancer cells and FasR-sensitive Jurkat T-cells. RESULTS: FasR was expressed in normal pancreas, in 14 of 14 pancreatic cancer cell lines, and in 3 of 3 surgical specimens. However, only 1 of 14 cancer cell lines expressed functional FasR when grown in monolayer, although 3 additional cell lines displayed functional FasR when cultured in suspension. Normal pancreas did not express FasL, whereas 14 of 14 cancer cell lines and 3 of 3 surgical specimens expressed FasL. FasL expressed by pancreatic cancer cells mediated killing of Jurkat T-cells in coculture assays (P < .005). CONCLUSIONS: These data suggest that pancreatic cancer cells have 2 potential mechanisms of evading Fas-mediated immune surveillance. A nonfunctional FasR renders them resistant to Fas-mediated apoptosis. The aberrant expression of functional FasL allows them to "counterattack" activated Fas-sensitive T-cells. Alone or in unison, these tumor escape mechanisms may contribute to the malignant and often rapid course of pancreatic cancer disease.

Successful adoptive cellular immunotherapy is dependent on induction of a host immune response triggered by cytokine (IFN-gamma and granulocyte/macrophage colony-stimulating factor) producing donor tumor-infiltrating lymphocytes.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and systemic low dose rIL-2 effectively eradicates pulmonary metastases of the murine MCA-105 sarcoma. We described earlier that host CD8+ T cells are critical for tumor eradication and that successful treatment is associated with production of high levels of IFN-gamma and granulocyte/macrophage (GM)-CSF by donor TIL in vitro. Here, we propose the mechanism through which adoptively transferred Thy-1.1+ TIL induce a host antitumor response in congenic Thy-1.2+ tumor-bearing mice. Donor Thy-1.1+ TIL were detected at the tumor site 12 h after transfer. These Thy-1.1+ cells produced IFN-gamma and GM-CSF in situ. The percentage of Thy-1.1+ TIL at the tumor site increased up to 16.4 +/- 4.9% 24 h after transfer but decreased to undetectable levels thereafter. In contrast, the percentages of host cells producing IFN-gamma and GM-CSF continued to increase at the tumor site. These increases were significantly higher in TIL + rIL-2-treated mice compared with untreated mice and rIL-2-treated mice 48 h after TIL transfer. The appearance of IFN-gamma+ and GM-CSF+ cells was followed by a large influx of host CD4+, CD8+, and Thy-1.2+ TIL and eventually by tumor eradication. This response was tumor specific since TIL obtained from MCA-205 did not induce high levels of IFN-gamma and GM-CSF and did not induce tumor eradication of MCA-105 tumor. Coinjection of Thy-1.1+ TIL and anti-IFN-gamma or anti-GM-CSF mAb significantly inhibited antitumor efficacy of the TIL + rIL-2 treatment. We conclude that successful adoptive immunotherapy in this model is mediated through cytokine production by adoptively transferred TIL that induce a host T cell-dependent antitumor response.

[Significance if ICAM-1 on lysis of human pancreatic carcinoma cells by autologous, cytotoxic T-lymphocytes]. / Die Bedeutung von ICAM-1 auf die Lyse von humanen Pankreaskarzinomzellen durch autologe, zytotoxische T-Lymphozyten.

Cytotoxic T-Lymphocytes were generated from tumor-infiltrating lymphocytes of a newly established pancreatic cancer cell line, which lysed autologous tumor cells significantly and HLA-class I restricted. ICAM-1+ tumor clones were significantly higher lysed than an ICAM-1- clone. Stimulation with IFN-gamma resulted in a higher expression of ICAM-1 and significantly higher lysis, which was inhibited by anti-ICAM-1 monoclonal antibodies.

Clone 10d/BM28 (CDCL1), an early S-phase protein, is an important growth regulator of melanoma.

Retinoic acid (RA) induces growth arrest and differentiation of many different tumor cells. RA activates RA receptors, which function as ligand-dependent transcriptional modulators. S91 murine melanoma cells stop proliferating and then reversibly differentiate into a melanocytic cell type after the administration of RA. The genetic changes that take place during this process serve as an excellent model for the etiology of melanoma. The use of subtractive hybridization techniques yielded several differentially expressed cDNAs that are associated with RA-induced growth arrest. One clone, cyclin D1, is repressed and is probably a differentiation marker. Two other cDNAs represent novel, RA-inducible genes. Expression of another cDNA, clone 10d, is strongly down-regulated. It is the homologue of the human gene BM28 (CDCL1) that is indispensable for entry into S phase and cell division. S91 cells that are permanently transfected with a plasmid that constitutively expresses clone 10d become significantly more resistant to RA, suggesting that repression of this gene is a critical event in RA-induced growth arrest. The use of reverse transcription-PCR for the detection of expression in human melanoma in vitro was performed to study the potential role of clone 10d/BM28 in this disease. It is expressed in 80% of melanoma cell lines but is virtually undetectable in primary melanocytes. The expression of BM28 is not regulated by RA in human, RA-resistant melanoma cells. These results suggest that clone 10d/BM28 functions as an important tumor cell growth promoter. The regulation of clone 10d may be directly mediated by RA receptors, and escape from negative regulation may, thus, contribute to the etiology of melanoma.

Generation of peptide-specific cytotoxic T lymphocytes using allogeneic dendritic cells capable of lysing human pancreatic cancer cells.

BACKGROUND: Dendritic cells (DCs) are potent antigen presenting cells (APCs), able to efficiently induce primary T cell-mediated responses to foreign antigens. In earlier studies we were able to identify a histocompatibility antigen (HLA)-A 2-restricted nine amino acid peptide (GP2, peptide 654-662) from the transmembrane portion of the protooncogene HER2/neu as a tumor-associated antigen (TAA) in human pancreatic cancer. METHODS: Peripheral blood mononuclear cells (PBMCs) of HLA-A2+ and HLA-A2 healthy volunteers were isolated. PBMCs were grown with initial anti-CD3, low-dose interleukin-2 (IL-2), and peptide-pulsed DC stimulation. T-cell lines were analyzed in functional studies. RESULTS: After 4 weeks, T-cell cultures were more than 50% CD8+. All peptide-pulsed T cells significantly lysed APC pulsed with the immunizing antigen in an HLA-A2 restricted fashion. Furthermore, HLA-A2+,HER2/neu+ human pancreatic cancer cells were lysed significantly higher than HLA-A2 HER2/neu+ pancreatic cancer cells. Transfection of an HLA-A2 pancreatic cancer cell line with the HLA-A2 gene resulted in a significantly higher lysis of the transfected cell line compared to the wild type. In HLA-A2+ pancreatic cancer targets, specific lysis was HLA-A2 restricted. CONCLUSION: The ability to use DCs for presentation of either tumor or peptide antigen in an HLA-restricted fashion to stimulate T-cell proliferation, as well as cytotoxicity, demonstrates the potential of this technology for future development of a pancreatic cancer vaccine.

The HER2/neu-derived peptide p654-662 is a tumor-associated antigen in human pancreatic cancer recognized by cytotoxic T lymphocytes.

The protooncogene HER2/neu encodes a 185-kDa transmembrane protein with extensive homology to the epidermal growth factor receptor. It is overexpressed in several human cancers of epithelial origin, such as pancreatic cancer. Previously, we demonstrated that cytotoxic T lymphocytes (CTL) derived from breast, ovarian, and non-small cell lung cancer recognized a peptide derived from HER2/neu. To evaluate whether this HLA-A2-binding peptide is a tumor-associated antigen (TAA) in pancreatic cancer, the ability of HER2/neu-reactive CTL to lyse human pancreatic carcinoma cells was tested. CTL were generated from tumor-associated T lymphocytes from HLA-A2+ HER2/neu+ breast and ovarian cancer patients. All CTL recognized autologous and allogeneic HER2/ neu+ tumor cells in an HLA-A2-restricted fashion. Furthermore, all CTL recognized p654-662 (GP2) derived from HER2/neu. These CTL also recognized HER2/neu+ pancreatic cancer cells in an HLA-A2-restricted fashion. HER2/neu+ HLA-A2- pancreatic cancer were not or only poorly lysed. Repeated stimulation of HLA-A2+ PBL from pancreatic cancer patients using the HER2/neu-derived peptide resulted in specific recognition of this peptide and, more importantly, HER2/neu+ pancreatic tumors in an HLA-A2-restricted fashion. Autologous HLA-A2+ fibroblasts or HLA-A2+ malignant melanoma cells were not recognized. HLA-A2- peptide-stimulated T lymphocytes showed no significant cytotoxicity. These results demonstrate that this HER2/neu-derived peptide is a shared TAA among several adenocarcinomas including pancreatic carcinoma, suggesting a common mechanism of recognition of these human tumors by T lymphocytes. The identification of the HER2/neu-derived peptide GP2 as a TAA in pancreatic cancer provides an opportunity for the design of novel immunotherapy and vaccine strategies.

Simultaneous production of T helper-1-like cytokines and cytolytic activity by tumor-specific T cells in ovarian and breast cancer.

Cytotoxic T-cell (CTL) cultures were generated from five ovarian cancer patients (OvCTL) and from three breast cancer patients (BrCTL). All CTL lines were T-cell receptor (TcR) alphabeta+ and predominantly CD8+ (73 +/- 13%). These CTL lines preferentially recognized autologous tumor cells in an HLA class I-restricted, and in part HLA-A2-restricted, manner. In addition, the CTL lines recognized allogeneic HLA-A2+ ovarian and breast tumor cells. Specific recognition was determined by T-cell-mediated cytotoxicity as well as cytokine release. Coculture of irradiated autologous tumor cells with OvCTL induced secretion of IFN-gamma, GM-CSF and TNF-alpha, but not IL-4, indicating a T helper-1-type response. Similar results were obtained when OvCTL and BrCTL were stimulated with histologically matched HLA-A2+ tumor cells. Also, BrCTL stimulated with HLA-A2+ but not HLA-A2- ovarian tumor cells produced significant levels of GM-CSF and TNF-alpha. Finally, the Her2/neu peptide p654-662, earlier identified as a tumor antigen in both ovarian and breast cancer, induced cytotoxicity as well as the specific release of IFN-gamma and TNF-alpha but not IL-4 by OvCTL and BrCTL. Thus, tumor-specific recognition by CTL was verified by cytotoxicity and cytokine release. The secretion of Th1-like cytokines as opposed to Th2-like cytokines suggest that therapeutically OvCTL and BrCTL could potentially enhance the endogenous immune response to tumor.

[Cytotoxic T-lymphocytes and the recognition of tumor antigens--an approach to "preventive tumor vaccination?"]. / Zytotoxische T-Lymphozyten und die Erkennung von Tumorantigenen--Ein Weg zur "Tumorschutzimpfung"?

In spite of modifications of the intra- and postoperative therapeutic strategy as well as new adjuvant protocols, most malignant diseases have a poor prognosis. This demonstrates the need for new approaches in tumor therapy. One alternative is adoptive immunotherapy (AIT). AIT is based on the observation that cytotoxic T-lymphocytes (CTL) may be generated with the ability to lyse autologous tumor cells in vitro and in vivo. Aim of current research activities is to determine specific tumorantigens and the clinical applicability of cell-mediated immunotherapy. The immunobiology of the cell-mediated immune response to cancer with focus on major-histocompatibility complex class I and CD8+ T cells is reviewed. Recent advances in the identification of tumor-associated and tumor-specific antigens were summarized. Previously performed clinical studies were reviewed. We examined the implications that the discovery of these antigens might have on the development of new anticancer vaccines.

Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro.

BACKGROUND: We investigated different culture conditions for tumor-infiltrating lymphocytes (TILs) with regard to proliferation, phenotypic changes, in vitro cytotoxicity, and in vivo therapeutic efficacy. METHODS: After enzymatic digestion of the murine fibrosarcoma, MCA-105, TIL cultures were initiated as pure lymphocyte (groups 1 and 2) or mixed lymphocyte/tumor suspensions (groups 3 and 4). Group I TILs were grown in culture medium containing 100 IU/ml recombinant interleukin-2 (rIL-2). Group 2 TILs were stimulated with solid-phase anti-CD3 monoclonal antibody (mAb) for 48 h and cultured in rIL-2 (100 IU/ml)-containing medium. Group 3, which consisted initially of a surplus of tumor cells, received the same treatment as group 2. Group 4 was also activated with anti-CD3 mAb and rIL-2 but was additionally restimulated weekly with irradiated tumor cells (TILs to tumor, 20:1). RESULTS: Groups 1 and 2 showed up to twofold higher increases in TIL numbers compared with groups 3 and 4 by the end of culture week 5. Although the original lymphocyte/tumor cell suspension consisted of 12.0 +/- 3.8% CD4+ T cells and 5.3 +/- 3.3% CD8+ T cells, all four TIL cultures showed approximately 80% CD8+ TILs and no CD4+ TILs by the end of culture week 4. In vitro cytotoxicity did not correlate with in vivo efficacy of the examined TIL cultures. By using the MCA-105 pulmonary metastases model in C57BL/6 mice, only suboptimal doses of TILs (2 x 10(6)) from group 4, which had been restimulated weekly with irradiated tumor, showed significant tumor eradication compared with all other treatment groups (p < 0.01). CONCLUSIONS: We conclude that in vitro tumor restimulation of TILs improves in vivo efficacy, most likely through the education of tumor-reactive T cells.

Vaccine therapy for cancer.

BACKGROUND: Tumor-specific cytotoxic T-lymphocytes (CTLs) can be isolated from the solid tumors, draining lymph nodes, metastatic effusions, and peripheral blood of cancer patients. Despite this evidence for a cell-mediated immune response to cancer, attempts at active specific immunotherapy using cancer vaccines have met with little success in clinical trials. METHODS: We have reviewed the immunobiology of the cell-mediated immune response to cancer by focusing on what is known about the major histocompatibility complex (MHC)-restricted interaction between tumor cells and CD8+ or CD4+ T-cells. In addition, we review the recent advances in the identification of tumor-associated antigens (TAAs) that are recognized by tumor-specific CTLs in melanoma and other cancers. In discussing these antigens, we highlight the recent identification of several MHC-restricted antigenic peptides that are recognized by CTLs from patients with melanoma and those with ovarian and breast cancer. We examine the implications that the discovery of these TAAs and peptides will have on the development of new anticancer vaccines. We review the most recent vaccine trials in melanoma and other cancers and focus on current concepts aimed at improving the therapeutic efficacy of future vaccines, including genetically engineered tumor cell vaccines. CONCLUSIONS: With the recent identification of several TAAs and antigenic peptide epitopes in melanoma and other cancers, immunotherapy researchers are now focusing on new strategies for the development of anticancer vaccines. As the repertoire of known TAAs increases and our understanding of the immunobiology of cell-mediated immunity to cancer improves, immunotherapists remain cautiously optimistic in their quest for effective cancer vaccines.

Tumor-specific and HLA-A2-restricted cytolysis by tumor-associated lymphocytes in human metastatic breast cancer.

A tumor-specific cytotoxic T lymphocyte (CTL) immune response has been well documented in melanoma, renal cell carcinoma, and ovarian cancer. Conflicting evidence exists regarding the existence of tumor-specific CTL populations in breast cancer. Tumor cells and tumor-associated lymphocytes (TAL) were isolated from the pleural effusions of six consecutive patients with metastatic breast cancer. After solid-phase anti-CD3 stimulation, TAL cultures were expanded with weekly autologous tumor stimulation and low-dose IL-2 for 3 wk. T cell populations were characterized using flow cytometric analysis and ranged from 49 to 91% CD8+, > 98% CD3+, and < 3% CD16+. Functionally, tumor-stimulated TAL showed tumor-specific recognition of autologous tumor cells (241 +/- 142 LU20/10(7)) and no detectable lysis of autologous fibroblasts, Daudi or K562. Cytotoxicity of TAL against HLA-A2+ allogeneic targets was significantly higher when compared with HLA-A2- tumor cell lines (127 +/- 76 vs 6 +/- 18 LU, p = 0.0001). This cytotoxicity against autologous and allogeneic tumor cells was blocked by anti-HLA-A2 mAb and cold HLA-A2+ targets in cold-target inhibition assays. TAL from all HLA-A2+ patients recognized GP2, a known, HER2/neu-derived tumor-associated peptide Ag that is HLA-A2 restricted. We have shown that TAL obtained from metastatic effusions of breast cancer patients contain lymphocytes that can recognize and lyse autologous and allogeneic tumor cells in a tumor-specific, HLA-A2-restricted fashion. In addition, tumor-specific TAL derived from breast cancer patients can selectively lyse HLA-A2+ pancreatic and ovarian tumor cell targets, suggesting a common HLA-A2-restricted tumor-associated Ag between these distinct epithelial cancers. Further elucidation of the cell-mediated immune response to breast cancer and the identification of shared TAA could result in the development of broadly applicable vaccine therapies for many cancers.