German G. Gomez

Antigenic Profiling of Glioma Cells to Generate Allogeneic Vaccines or Dendritic Cell–Based Therapeutics

Purpose: Allogeneic glioma cell lines that are partially matched to the patient at class I human leukocyte antigen (HLA) loci and that display tumor-associated antigens (TAA) or antigenic precursors [tumor antigen precursor proteins (TAPP)] could be used for generating whole tumor cell vaccines or, alternatively, for extraction of TAA peptides to make autologous dendritic cell vaccines. Experimental Design: Twenty human glioma cell lines were characterized by molecular phenotyping and by flow cytometry for HLA class I antigen expression. Twelve of the 20 cell lines, as well as analyses of freshly resected glioma tissues, were further characterized for protein and/or mRNA expression of 16 tumor antigen precursor proteins or TAA. Results: These 20 human glioma cell lines potentially cover 77%, 85%, and 78% of the U.S. Caucasian population at HLA-A, HLA-B, and HLA-C alleles, respectively. All cells exhibited multiple TAA expressions. Most glioma cells expressed antigen isolated from immunoselected melanoma-2 (Aim-2), B-cyclin, EphA2, GP100, β1,6-N-acetylglucosaminyltransferase V (GnT-V), IL13Rα2, Her2/neu, hTert, Mage, Mart-1, Sart-1, and survivin. Real-time PCR technology showed that glioblastoma specimens expressed most of the TAA as well. Tumor-infiltrating lymphocytes and CD8+ CTL killed T2 cells when loaded with specific HLA-A2+ restricted TAA, or gliomas that were both HLA-A2+ and also positive for specific TAA (Mart-1, GP100, Her2/neu, and tyrosinase) but not those cells negative for HLA-A2 and/or lacking the specific epitope. Conclusions: These data provide proof-in-principle for the use of allogeneic, partially HLA patient–matched glioma cells for vaccine generation or for peptide pulsing with allogeneic glioma cell extracts of autologous patient dendritic cells to induce endogenous CTL in brain tumor patients.

Human Alloreactive CTL Interactions with Gliomas and with Those Having Upregulated HLA Expression from Exogenous IFN-γ or IFN-γ Gene Modification

By flow cytometry, a panel of 18 primary glioma cell explants exhibited high expression of class I HLA-A, B, C, but class II HLA-DR expression was absent. Freshly isolated normal brain cells displayed little or no HLA antigens. Alloreactive cytotoxic T lymphocytes (aCTL), sensitized to the HLA of the patient, were generated in a one-way mixed lymphocyte response (MLR). The specificity of aCTL was confirmed to be to target cells (patient glioma cells or lymphoblasts) expressing the relevant HLA antigens. However, nontumor patient-specific aCTL did not lyse normal brain cells. Titration of antibodies to HLA class I into cytotoxicity assays blocked lysis of gliomas by aCTL, confirming aCTL T cell receptor (TCR) interactions with the class I antigen on gliomas. Furthermore, aCTL interactions with glioma cells caused their apoptosis. Coincubations of aCTL with gliomas resulted in upregulated cytokine secretion. Importantly, dexamethasone, an immunosuppressive steroid used for brain edema, did not affect aCTL lytic function against tumor, indicating that steroid-dependent patients may benefit from the immunotherapy. We also explored the use of interferon-gamma (IFN-gamma) to increase aCTL tumor recognition. Coincubation of gliomas with exogenous IFN-gamma (500 U/ml, 48 h) caused a 3-fold upregulation of HLA class I and a slight induction of class II antigen expression. Gene-modified glioma cells producing IFN-gamma similarly displayed upregulated HLA expression. Glioma cells incubated with exogenous IFN-gamma or IFN-gamma-transduced glioma cells were more susceptible to lysis by aCTL than their parental counterparts, thus supporting the concept of combining IFN-gamma cytokine gene therapy with adoptive aCTL immunotherapy for brain tumor treatment.

Cellular and functional characterization of immunoresistant human glioma cell clones selected with alloreactive cytotoxic t lymphocytes reveals their up-regulated synthesis of biologically active TGF-β

Two immunoresistant (IR) glioma cell variants, 13-06-IR29 and 13-06-IR30, were cloned from 13-06-MG glioma cell populations after receiving continuous immunoselective pressure from multiple alloreactive cytotoxic T lymphocyte (aCTL) preparations. Reapplication of aCTL immunoselective pressure to the IR clones, displaying a partial regain in sensitivity to aCTL after removal of the selective pressure, restored the resistance. The IR variants exhibited cross-resistance to non-human leukocyte antigen (HLA)-restricted effector cells and γ-irradiation, but not to carmustine. The IR clones were characterized for factors that might contribute to the immunoresistance. The aCTL adhesion to extracellular matrix extracts derived from either the IR clones or the parental cells was similar and not impaired. Furthermore, aCTL binding to parental cells and IR clones was equal. Down-regulation of the cell recognition molecules, class I HLA or intercellular adhesion molecule-1 (ICAM-1), that would inhibit their recognition by aCTL was not observerd on the IR clones. The down-regulation of Fas by the IR clones correlated with their resistance to FasL-induced apoptosis. HLA-G or FasL that might provide an immunotolerant environment or provide a means of counterattack to aCTL, respectively, were not associated with the IR phenotype. The aCTL, coincubated with the IR clones and parental cells, displayed up-regulation of multiple secreted cytokines. A significant up-regulation of bioactive transforming growth factor (TGF)-β was observed in the IR clones compared with the parental cells. These data suggest that increased secretion of bioactive TGF-β may inhibit aCTL lysis of the IR clones. Disruption of the TGF-β signaling pathway may circumvent the resistance.

Effects of IFN-γ and Interleukin-1β on Major Histocompatibility Complex Antigen and Intercellular Adhesion Molecule-1 Expression by 9L Gliosarcoma: Relevance to Its Cytolysis by Alloreactive Cytotoxic T Lymphocytes

To enhance the efficacy of cellular immunotherapy for gliomas, we tested the concept of using proinflammatory cytokine treatment with interferon-γ (IFN-γ) or interleukin-1β (IL-1β) or both to render glioma cells more susceptible to cytolysis by alloreactive cytotoxic T lymphocytes (aCTL). The cytokines, separately or in combination, were able to upregulate major histocompatibility complex (MHC) class I antigen or intercellular adhesion molecule-1 (ICAM-1) on Fischer rat 9L gliosarcoma cells. 9L cells were incubated in vitro for 24, 48, or 72 h with varying concentrations of rat IFN-γ (0-2000 U/ml) or recombinant human IL-1 (rHUIL-1) (0-1000 U/ml) or both. By 48 h, IFN-γ (500 U/ml) maximally induced the percentage of positive expressing cells and the relative antigen density of MHC class I and ICAM-1 on 9L cells, whereas IL-1 induced only ICAM-1 expression. Simultaneous incubation of IL-1 with IFN-γ did not further affect the induction of class I on 9L cells more than that achieved with IFN-γ alone. 9L cel...

γ Interferon transduced 9L gliosarcoma. Cytokine gene therapy and its relevance to cellular therapy with alloreactive cytotoxic T lymphocytes

SummaryIn earlier studies, we demonstrated that intratumoral infusions of alloreactive cytotoxic T lymphocytes (aCTL), sensitized to the major histocompatibility complex (MHC) antigens of the host, effectively retarded the intracranial growth of Fischer 9L gliosarcoma. We further demonstrated that continuousin vitro exposure to gamma-interferon (γIFN) upregulates MHC on 9L gliosarcoma cells and that they were better targets of anti-Fischer aCTL. We hypothesized that the efficacy of cellular therapy with aCTL could be further improved byin situ transduction of the tumor with retroviral vectors coding forγIFN, which would generate continuous secretion of the cytokine and maintain upregulated MHC expression by the tumor cells. 9L gliosarcoma and Herpes simplex virus thymidine kinase (tk) transductants of those cells were transduced with a retrovirus carrying the murineγIFN gene. By limiting dilution, clones of these cells, designated 9Lγ7, 9Lγtk8, and 9Lγtk10, which produced similar levels ofγIFN (383–411 ngγIFN/106 cells/24h) were isolated. The production ofγIFN by one clone, 9Lγ7, was stable when monitored over 6 weeksin vitro. The clones also demonstrated upregulated MHC class I expression, and the tk-transduced clones maintained their sensitivity to ganciclovir. Compared to the wildtype cells, 9Lγ7 had approximate 6- and 1.5-fold increases in the relative antigen densities of MHC I and II, respectively. Addition of exogenousγIFN to 9Lγ7 cultures did not significantly increase the MHC expression. In cytotoxicity assays, 9Lγ7 cells, or 9Lγ7 incubated with exogenousγIFN, were better targets of aCTL than the parental 9L cells. The growth rate of 9Lγ-transduced cells was decreased compared to the wildtype cells bothin vitro andin vivo. Proliferation studies with transwell plated 9L, 9Lγ7, and 9Lγtk10 cells in various combinations revealed that the secreted cytokine itself caused a decrease in proliferation. However, the transduced cells exhibited a much reduced growth rate, which likely was a consequence of redirected metabolic activity of the cells.In vivo growth of the 9L and 9Lγ7 tumors in rat brains given identical inoculums similarly demonstrated significantly reduced 9Lγ7 tumor volumes at various timepoints, indicative of slower growth of theγIFN-producing tumors.