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HER-2/neu is a self-antigen expressed by tumors and nonmalignant epithelial tissues. The possibility of self-tolerance to HER-2/neu-derived epitopes has raised questions concerning their utility in antitumor immunotherapy. Altered HER-2/neu peptide ligands capable of eliciting enhanced immunity to tumor-associated HER-2/neu epitopes may circumvent this problem. The human CTL peptide HER-2/neu (435-443) [hHER-2(9(435))] represents a xenogeneic altered peptide ligand of its mouse homologue, differing by one amino acid residue at position 4. In contrast to mHER-2(9(435)), vaccination of HLA-A*0201 transgenic (HHD) mice with hHER-2(9(435)) significantly increased the frequency of mHER-2(9(435))-specific CTL and also induced strong protective and therapeutic immunity against the transplantable ALC tumor cell line transfected to coexpress HLA-A*0201 and hHER-2/neu or rHER-2/neu. Similar results were also obtained with wild-type C57BL/6 mice inoculated with HER-2/neu transfectants of ALC. Adoptive transfer of CD8(+) CTL from mice immunized with hHER-2(9(435)) efficiently protected naive syngeneic mice inoculated with ALC tumors. In conclusion, our results show that HER-2(9(435)) serves as a tumor rejection molecule. They also propose a novel approach for generating enhanced immunity against a self-HER-2/neu CTL epitope by vaccinating with xenogeneic altered peptide ligands and provide useful insights for the design of improved peptide-based vaccines for the treatment of patients with HER-2/neu-overexpressing tumors.

IL-21 plays a role in the proliferation and maturation of NK cells developed from hematopoietic stem cells. In this study, we found that IL-21, in the presence of physiological concentration of hydrocortisone (HC), has a significant impact on the functions of NK cells derived from umbilical cord blood (CB) populations. We demonstrate that IL-21, in combination with Flt3-ligand, IL-15 and HC, induces high proliferative responses and, apart from enhancing NK-mediated cytotoxicity, it also induces a significant increase in lymphokine-activated killer activity of CB/CD34+-derived CD56+ cells. In addition, IL-21 induced changes in the CD56+ cell cytokine secretion profile. Thus, we observed increased levels of IL-10 and granulocyte macrophage colony-stimulating factor, whereas tumor necrosis factor-alpha levels decreased. IFN-gamma production was also modified by IL-21, depending on the presence or absence of IL-18. CB/CD34+ cells did not express the IL-21R ex vivo, but receptor expression was induced during their commitment to differentiation into CD56+ cells. Our data ascribe to IL-21 an essential role on NK cell development and function under conditions similar to the in vivo CB microenvironment.

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination.

We measured absolute counts (cells/microl) of Calcein-AM stained target cells that remained viable [i.e. not permeable to the viability probe 7-AAD (7-Amino Actinomycin D)] following a four-hour incubation with effectors [NK, CTL (cytotoxic T lymphocytes)] without using beads or standards. The absolute counts were evaluated by a Cytoron Absolute (Ortho) flow cytometry apparatus. Median triplicate counts were compared at 0 and 4h, in single targets and increased effector/target ratios. The Coefficient of Variation (CV) of the cell concentration (cells/microl) at the beginning of the experiment was below 6%. The % changes of viable target counts were correlated to the effector/target ratios by linear regression. The methodology was applied in pairs: 17 for allogenic stem cell transplantation from unrelated donors, 3 of healthy unrelated individuals, 10 for measuring NK activity and 7 autologous. In 15/20 allogenic MLC (mixed lymphocytes culture) and for all NK assays the cytotoxicity was positive (p < 0.05, r2 > 0.9) while in 5/20 allogeneic MLC and in all autologous MLC the outcome was negative (p > 0.05, r2 < 0.4). The proposed method offers the advantage of combining absolute counts with flow cytometry analysis of viable targets, assessment of cell behavior during the cytotoxic phenomenon, the use of a small amount of cells and excellent sensitivity. Our method can estimate frequencies higher than 1:100 for CTL assays.

Background: We recently demonstrated that supernatants from cultures of peripheral blood mononuclear cells (PBMC) activated with anti-CD3-specific antibody (ACD3S) can induce, upon brief exposure, tumor-reactive lymphocytes in cancer patients. Here, we report that ACD3S can also induce rapid and stable maturation of dendritic cells (DC) which can be used as antigen presenting cells in in vitro protocols and for cancer immunotherapy in vivo., Materials and Methods: A short (4-day) priming of CD14+ monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) followed by only a 24 hour-incubation in ACD3S, is sufficient to generate fully mature and stable DC., Results: These DC (i) stimulated strong T cell proliferative responses in the mixed lymphocyte reaction, (ii) when pulsed with unfractionated peptides from autologous tumor membrane extracts activated CD4+ T cells which proliferated in response to the autologous tumor and CD8+ cytotoxic T cells (CTL) which specifically lyse autologous tumor targets and (iii) produced high levels of IL-12., Conclusion: ACD3S-treated DC are functionally superior to monocyte-conditioned medium (MCM)-treated DC generated under the same short-term protocol and as efficient as DC induced by the standard 10-day protocol. Our data present an efficient and effective method for generating in a very short period of time, highly mature and functionally competent DC.