Alessandro D. Santin

Induction of ovarian tumor-specific CD8+ cytotoxic T lymphocytes by acid-eluted peptide-pulsed autologous dendritic cells

Objective To evaluate the potential of dendritic cells pulsed with acid-eluted peptides derived from autologous ovarian cancer cells for eliciting a tumor-specific cytotoxic T cell response in women with advanced ovarian cancer. Methods CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous ovarian tumor peptide-pulsed dendritic cells were tested for their ability to induce an HLA class I–restricted cytotoxic T lymphocyte response against autologous tumor cells. To correlate cytotoxic activity by cytotoxic T lymphocytes with T cell phenotype, we used two-color flow cytometric analysis of surface markers and intracellular cytokine expression (interferon-γ versus interleukin-4). Results CD8+ cytotoxic T lymphocyte responses against autologous ovarian tumor cells were elicited in three consecutive women who had advanced ovarian cancer. Although cytotoxic T lymphocyte populations from all women expressed strong cytolytic activity against autologous tumor cells, they did not lyse autologous lymphoblasts or Epstein-Barr virus-transformed cell lines, and they showed negligible cytotoxicity against the natural killer-sensitive cell line K-562. Cytotoxicity against the autologous tumor cells was significantly inhibited by anti-HLA class I (W6/32) and anti-HLA-A2 (BB7-2) monoclonal antibodies. CD8+ cytotoxic T lymphocytes expressed variable levels of CD56 and preferentially expressed interferon-γ rather than interleukin-4. Conclusions Peptide-pulsed dendritic cells induced specific CD8+ cytotoxic T lymphocytes that killed autologous tumor cells from women with advanced ovarian cancer. This finding might contribute to the development of active or adoptive immunotherapy for residual or resistant ovarian cancer after standard surgery and cytotoxic treatment.

Effects of irradiation on the expression of major histocompatibility complex class I antigen and adhesion costimulation molecules ICAM-1 in human cervical cancer

PURPOSE We initiated studies to analyze the effects of high doses of gamma irradiation on the surface antigen expression of MHC Class I, Class II, and ICAM-1 on human cervical carcinoma cell lines. METHODS AND MATERIALS The expression of surface antigens (MHC Class I, Class II, and ICAM-1) was evaluated by FACS analysis on two cervical cell lines at different time points, following their exposure to high doses of gamma irradiation (i.e., 25.00, 50.00, and 100.00 Gy). RESULTS The CaSki and SiHa cervical cancer cells we analyzed in this study expressed variable levels of MHC Class I and ICAM-1 antigens, while Class II surface antigens were not detectable. Whereas irradiation doses of 25.00 Gy were not sufficient to totally block cell replication in both cell lines, exposure to 50.00 or 100.00 Gy was able to completely inhibit cell replication. Range doses from 25.00 to 100.00 Gy significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation but were unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). Importantly, such upregulation was shown to be dose dependent, with higher radiation doses associated with increased antigen expression. Moreover, when the kinetic of this upregulation was studied after 2 and 6 days after irradiation, it was shown to be persistent and lasted until all the cells died. CONCLUSIONS These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy.

The effects of irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical cancer.

PURPOSE Studies were designed to analyse the effects of high doses of gamma-irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical carcinoma cell lines. MATERIALS AND METHODS The expression of heat shock protein gp96 was evaluated at the transcriptional (Northern blot) and post-transcriptional levels (Western blot) in two human cervical carcinoma cell lines following exposure to high doses of gamma-irradiation. RESULTS Doses of gamma-irradiation ranging from 25 to 100 Gy significantly and consistently increased the expression of heat shock protein gp96 on CaSki and HT-3 cervical cancer cells. The increase in the amount of protein was due to transcriptional up-regulation of this gene. Radiation doses unable to inhibit completely cell replication in the totality of tumour cells (i.e. 25 Gy), as well as higher (fully lethal) doses of irradiation (i.e. 50 to 100 Gy), were shown to up-regulate significantly the expression of heat shock protein gp96 mRNA in a dose-dependent manner. CONCLUSIONS Recently, gp96 molecules have been implicated in the presentation of endogenous and viral antigens. A number of key elements in this pathway, including major histocompatibility complex (MHC) class I molecules as well as adhesion/co-stimulation molecules such as ICAM-1, are known to be sensitive to irradiation effects. The results show that radiation can also increase the expression of other immunologically important cell molecules such as a tumour rejection antigen (heat shock protein gp96) in human cervical cancer. Such findings may partially explain the increased immunogenicity of tumour cells following irradiation and further support a role for local radiation therapy as a powerful biologic response modifier.

Secretion of vascular endothelial growth factor in adenocarcinoma and squamous cell carcinoma of the uterine cervix.

OBJECTIVE To determine whether major differences in vascular endothelial growth factor secretion exist between adenocarcinomas of the uterine cervix compared with squamous cell carcinomas. METHODS The secretion of vascular endothelial growth factor by eight fresh cervical cancer cell preparations (four adenocarcinomas and four squamous cell carcinomas) and four established squamous cell lines was evaluated using a sensitive enzyme-linked immunosorbent assay in vitro. RESULTS All cervical tumors secreted significant amounts of vascular endothelial growth factor, and no significant differences between fresh and established squamous cell lines were detectable. In contrast, a highly significant difference in vascular endothelial growth factor secretion was noted between fresh adenocarcinomas (mean = 2712, range between 1700 to 3500 pg/mL/10(5) cells/48 hours) when compared with fresh squamous (mean = 575, range between 200 to 950 pg/mL/10(5) cells/48 hours) or established squamous cervical carcinoma cell lines (mean = 712, range between 400 to 1000 pg/mL/10(5) cells/48 hours) (F-test, P< or =.001). CONCLUSION These data strongly suggest that major differences in the secretion of vascular endothelial growth factor exist between squamous cell carcinoma and adenocarcinomas of the uterine cervix. Therefore, at least some of the differences in the natural biologic behavior of these two histologic types of cervical cancer, including the propensity for earlier lymphatic and hematogenous metastasis as well as the lower response to radiation treatment, could be related to major differences in the secretion of this powerful angiogenic and immunosuppressive cytokine.

Development and therapeutic effect of adoptively transferred T cells primed by tumor lysate-pulsed autologous dendritic cells in a patient with metastatic endometrial cancer

We describe a 65-year-old woman with a large surgically unresectable and chemoresistant liver metastasis of endometrial carcinoma who was treated by infusion with peripheral blood T cells stimulated with tumor lysate-pulsed autologous dendritic cells (DC). Extensive in vitro characterization of the DC-activated T cells included phenotypic analysis, cytotoxicity, and intracellular cytokine secretion. High cytotoxicity was observed against autologous tumor cells, but not against NK-sensitive K562 cells, autologous Con-A lymphoblasts, or autologous Epstein-Barr virus-transformed lymphoblastoid cells. Blocking studies demonstrated that lytic activity was HLA class I restricted. Two-color flow cytometric analysis revealed that a significant proportion of CD8+ T cells was also CD56+, and analysis of intracellular IFN-γ and IL-4 expression suggested a type 1 cytokine bias. The patient was treated by three infusions of tumor-specific T cells at 3- to 4-week intervals, and in vivo distribution of the T cells was followed by 111In oxine labeling and serial gamma camera imaging. Tumor localization and accumulation of labeled lymphocytes was consistently detected at serial time points following each injection. However, deep infiltration of the large tumor mass by activated T cells was minimal, as evaluated in 3 dimensions by single photon emission computerized tomography (SPECT) imaging. Transient serum increases of the tumor marker lactate dehydrogenase (LDH), were detectable after each injection. Similar posttreatment elevations were seen for serum uric acid and potassium. Clinically, stabilization of the large liver metastasis was obtained during treatment. Collectively, these results indicate that tumor-specific CD8+ cytotoxic T-cell responses can be generated in patients with endometrial cancer, and suggest that T-cell immunotherapy may be of therapeutic value in patients harboring metastatic disease.