A urokinase receptor-Bim signaling axis emerges during EGFR inhibitor resistance in mutant EGFR glioblastoma.

EGFR is the most common genetically altered oncogene in glioblastoma (GBM), but small-molecule EGFR tyrosine kinase inhibitors (TKI) have failed to yield durable clinical benefit. Here, we show that in two novel model systems of acquired resistance to EGFR TKIs, elevated expression of urokinase plasminogen activator (uPA) drives signaling through the MAPK pathway, which results in suppression of the proapoptotic BCL2-family member protein BIM (BCL2L11). In patient-derived GBM cells and genetic GBM models, uPA is shown to suppress BIM levels through ERK1/2 phosphorylation, which can be reversed by siRNA-mediated knockdown of uPA. TKI-resistant GBMs are resensitized to EGFR TKIs by pharmacologic inhibition of MEK or a BH3 mimetic drug to replace BIM function. A link between the uPA-uPAR-ERK1/2 pathway and BIM has not been previously demonstrated in GBM, and involvement of this signaling axis in resistance provides rationale for a new strategy to target EGFR TKI-resistant GBM.

Suppression of microRNA-9 by mutant EGFR signaling upregulates FOXP1 to enhance glioblastoma tumorigenicity.

The EGF receptor (EGFR) is amplified and mutated in glioblastoma, in which its common mutation (ΔEGFR, also called EGFRvIII) has a variety of activities that promote growth and inhibit death, thereby conferring a strong tumor-enhancing effect. This range of activities suggested to us that ΔEGFR might exert its influence through pleiotropic effectors, and we hypothesized that microRNAs might serve such a function. Here, we report that ΔEGFR specifically suppresses one such microRNA, namely miR-9, through the Ras/PI3K/AKT axis that it is known to activate. Correspondingly, expression of miR-9 antagonizes the tumor growth advantage conferred by ΔEGFR. Silencing of FOXP1, a miR-9 target, inhibits ΔEGFR-dependent tumor growth and, conversely, de-repression of FOXP1, as a consequence of miR-9 inhibition, increases tumorigenicity. FOXP1 was sufficient to increase tumor growth in the absence of oncogenic ΔEGFR signaling. The significance of these findings is underscored by our finding that high FOXP1 expression predicts poor survival in a cohort of 131 patients with glioblastoma. Collectively, these data suggest a novel regulatory mechanism by which ΔEGFR suppression of miR-9 upregulates FOXP1 to increase tumorigenicity.

Therapeutic resistance in cancer: microRNA regulation of EGFR signaling networks.

Receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) regulate cellular homeostatic processes. EGFR activates downstream signaling cascades that promote tumor cell survival, proliferation and migration. Dysregulation of EGFR signaling as a consequence of overexpression, amplification and mutation of the EGFR gene occurs frequently in several types of cancers and many become dependent on EGFR signaling to maintain their malignant phenotypes. Consequently, concerted efforts have been mounted to develop therapeutic agents and strategies to effectively inhibit EGFR. However, limited therapeutic benefits to cancer patients have been derived from EGFR-targeted therapies. A well-documented obstacle to improved patient survival is the presence of EGFR-inhibitor resistant tumor cell variants within heterogeneous tumor cell masses. Here, we summarize the mechanisms by which tumors resist EGFR-targeted therapies and highlight the emerging role of microRNAs (miRs) as downstream effector molecules utilized by EGFR to promote tumor initiation, progression and that play a role in resistance to EGFR inhibitors. We also examine evidence supporting the utility of miRs as predictors of response to targeted therapies and novel therapeutic agents to circumvent EGFR-inhibitor resistance mechanisms.

A kinome-wide RNAi screen in Drosophila Glia reveals that the RIO kinases mediate cell proliferation and survival through TORC2-Akt signaling in glioblastoma.

Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK) and Pi-3 kinase (PI3K) signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.

Implementing preclinical study findings to protocol design: translational studies with alloreactive CTL for gliomas.

We are accruing patients to a Phase I dose escalation cellular therapy trial (www.clinicaltrials.gov, NCT01144247) involving intratumoral placement of alloreactive cytotoxic T lymphocytes (alloCTL) for recurrent gliomas. The trial is being conducted to confirm the findings of a prior pilot study that indicated this adjuvant therapy may be beneficial in extending survival of recurrent WHO grade III gliomas. To reduce costs of the cellular therapy, we tested a number of synthetic tissue culture media and found the AIM-V growth medium superior for their growth. We also moved the production of the alloCTL from artificial capillary systems to less expensive tissue culture bags. To standardize alloCTL infusates used for therapy, release criteria of ≥60% CD3+ and ≥60% viability were established that consistently translated to a 4 hr cytotoxicity of ≥30% at a 30:1 effector to target ratio. To allow time for completion of quality control testing and transport to the infusion site, we determined that 30,000 IU of human recombinant Interleukin-2 in the cellular infusates sufficiently retained cell viability and cytotoxicity to allow a 10 hr expiration time to be placed on the infusates. We identified a cytotoxic T cell subset, CD3+/CD8+/CD69+, that demonstrated upregulated IFN-γ production upon exposure to relevant target cells. The phenotypic identification of this T cell subset was indicative of robust in vitro cytotoxic function and thus will be followed to determine if it correlates with patient immune response to treatment. Finally, other therapeutic agents routinely used for glioma treatment were integrated into an analysis of alloCTL cytotoxic functionality. Temozolomide and bevacizumab do not adversely affect cytotoxic function of the alloCTL in the short-term, thus providing rationale for further investigating combinatorial chemo-immunotherapy for gliomas.

Immunoresistant human glioma cell clones selected with alloreactive cytotoxic T lymphocytes: downregulation of multiple proapoptotic factors.

We previously reported the cellular, functional and cytogenetic characterization of immunoresistant (IR) 13-06-IR29 and 13-06-IR30 human glioma cell clones isolated after immunoselection with alloreactive cytotoxic T lymphocytes (aCTL). Relative to the 13-06-MG parental cells, both clones resisted aCTL lysis at multiple effector to target ratios; the resistant phenotype was maintained for 13-41 cell doublings after cloning and when selective pressure was removed; cross-resistance to other inducers of apoptosis/cell death was also observed (Gomez et al, 2006; Gomez and Kruse, 2007). In this study we further characterize the IR clones for factors that may contribute to the resistance. Data obtained by in-vitro quantitative morphologic and 7-amino actinomycin D flow cytometric assays revealed reduced apoptotic cell death when IR clones were coincubated with aCTL, relative to the parental cells. Since changes in apoptosis were observed, we examined the expression patterns of apoptosis-related genes in several extracts of parental cells and IR clones using pathway-specific cDNA microarray analysis. In general, the apoptotic factors were downregulated in the IR clones. From three separate extracts analyzed separately on microarrays, three factors, ATM, caspases 3 and 8, were statistically downregulated in both IR clones. Immunoblotting of the proteins confirmed the findings. Therefore, a possible mechanism for immunoresistance in gliomas may be achieved by the downregulation of one or more genes in the apoptotic pathway.

siRNA Down-regulation of the PATZ1 Gene in Human Glioma Cells Increases Their Sensitivity to Apoptotic Stimuli.

The PATZ1 gene encodes a transcription factor that belongs to the BTB/POZ group of transcriptional regulators and has been implicated as a transcriptional repressor. We cloned cDNA from glioma cell lines and found they expressed transcript variant 2 of PATZ1. We designed a specific siRNA against PATZ1 and showed that this siRNA, but not a control randomized siRNA, reduced PATZ1 expression in glioma cells as determined by quantitative PCR. In a panel of human glioma cell lines incubated with proapoptotic FasL, those transfected with PATZ1 siRNA displayed reduced cell numbers by the MTT colorimetric assay, relative to those transfected with randomized siRNA. Further studies showed that in 10-08-MG, U-251MG, U-87MG, and T98G cells PATZ1 siRNA significantly increased apoptosis in response to incubation with soluble FasL, as shown by a morphologic acridine orange/ethidium bromide apoptotic assay. Using an apoptosis specific cDNA microarray we further demonstrated that down-regulation of PATZ1 by siRNA resulted in the upregulation of death receptor pro-apoptotic genes including caspase 8 and Death Receptor 5 (DR5) in U-373MG cells. Since DR5 is the receptor for TRAIL we tested whether PATZ1 downregulation also sensitized cells to TRAIL-induced apoptosis and found that PATZ1 siRNA, but not control siRNA, sensitized U-251MG and T98G glioma cells to TRAIL-induced apoptosis. Altogether, these data demonstrate a previously unknown role for the transcription factor PATZ1 in conferring resistance to apoptosis and indicate that modulation of PATZ1 expression may be a therapeutic strategy for gliomas.

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-beta.

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 gamma-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 observed 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)-beta was observed in the IR clones compared with the parental cells. These data suggest that increased secretion of bioactive TGF-beta may inhibit aCTL lysis of the IR clones. Disruption of the TGF-beta signaling pathway may circumvent the resistance.

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, beta1,6-N-acetylglucosaminyltransferase V (GnT-V), IL13Ralpha2, 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.

Mechanisms of malignant glioma immune resistance and sources of immunosuppression.

High grade malignant gliomas are genetically unstable, heterogeneous and highly infiltrative; all characteristics that lend glioma cells superior advantages in resisting conventional therapies. Unfortunately, the median survival time for patients with glioblastoma multiforme remains discouraging at 12-15 months from diagnosis. Neuroimmunologists/oncologists have focused their research efforts to harness the power of the immune system to improve brain tumor patient survival. In the past 30 years, small numbers of patients have been enrolled in a plethora of experimental immunotherapy Phase I and II trials. Some remarkable anecdotal responses to immune therapy are evident. Yet, the reasons for the mixed responses remain an enigma. The inability of the devised immunotherapies to consistently increase survival may be due, in part, to intrinsically-resistant glioma cells. It is also probable that the tumor compartment of the tumor-bearing host has mechanisms or produces factors that promote tumor tolerance and immune suppression. Finally, with adoptive immunotherapy of ex vivo activated effector cell preparations, the existence of suppressor T cells within them theoretically may contribute to immunotherapeutic failure. In this review, we will summarize our own studies with immunotherapy resistant glioma cell models, as well as cover other examined immunosuppressive factors in the tumor microenvironment and immune effector cell suppressor populations that may contribute to the overall immune suppression. An in-depth understanding of the obstacles will be necessary to appropriately develop strategies to overcome the resistance and improve survival in this select population of cancer patients.

Isolation of immunoresistant human glioma cell clones after selection with alloreactive cytotoxic T lymphocytes: cytogenetic and molecular cytogenetic characterization.

Intratumoral heterogeneity and genetic instability within gliomas may allow intrinsically immunoresistant (IR) cells to escape alloreactive cytotoxic T lymphocyte (aCTL) cellular immunotherapy. The potential existence of aCTL-resistant variants prompted us to investigate whether cellular immunotherapy resistant glioma models could be isolated. To generate the models, repeated intermittent or continuous selective pressure (ISP or CSP) with multiple aCTL populations was applied to a low-passage glioblastoma cell explant, 13-06-MG, obtained from a patient at initial diagnosis. IL-6 and IL-8 secretion was greater in coincubates of aCTL cells with 13-06-ISP and 13-06-CSP immunoselected cells than those with 13-06-MG parental cells. Initially, the immunoselected cells were less sensitive to aCTL lysis; however, the reduced aCTL-sensitivity was not maintained upon further selection. We therefore isolated IR clones from continuously immunoselected cells (13-06-CSP). The frequency of IR clones was 1-6 cells per 10,000 immunoselected cells. Two clones selected for further study, 13-06-IR29 and 13-06-IR30, resisted aCTL lysis in the absence of immunoselective pressure. Cytogenetic analyses revealed structural anomalies and genomic imbalances unique to the IR clones. Based on these findings, a hypothetical model is proposed that traces the origin of the IR clones to a clonal variant within the 13-06-CSP and 13-06-MG populations.

Catalytic nucleic acid enzymes for the study and development of therapies in the central nervous system: Review Article.

Nucleic acid enzymes have been used with great success for studying natural processes in the central nervous system (CNS). We first provide information on the structural and enzymatic differences of various ribozymes and DNAzymes. We then discuss how they have been used to explore new therapeutic approaches for treating diseases of the CNS. They have been tested in various systems modeling retinitis pigmentosum, proliferative vitreoretinopathy, Alzheimer's disease, and malignant brain tumors. For these models, effective targets for nucleic acid enzymes have been readily identified and the rules for selecting cleavage sites have been well established. The bulk of studies, including those from our laboratory, have emphasized their use for gliomas. With the availability of multiple excellent animal models to test glioma treatments, good progress has been made in the initial testing of nucleic acid enzymes for brain tumor therapy. However, opportunities still exist to significantly improve the delivery and efficacy of ribozymes to achieve effective treatment. The future holds significant potential for the molecular targeting and therapy of eye diseases, neurodegenerative disorders, and brain tumors with these unique treatment agents.

Interactions of the allogeneic effector leukemic T cell line, TALL-104, with human malignant brain tumors.

TALL-104 is a human leukemic T cell line that expresses markers characteristic of both cytotoxic T lymphocytes and natural killer cells. TALL-104 cells are potent tumor killers, and the use of lethally irradiated TALL-104 as cellular therapy for a variety of tumors has been explored. We investigated the interactions of TALL-104 cells with human brain tumor cells. TALL-104 cells mediated increased lysis of a panel of brain tumor cells at low effector-to-target ratios over time. We obtained evidence that TALL-104 cells injured glioma cells by both apoptotic and necrotic pathways. A 7-amino actinomycin D flow cytometry assay revealed that the percentages of both apoptotic and necrotic glioma cells increased after TALL-104 cell/glioma cell coincubations. Fluorescent microscopy studies and a quantitative morphologic assay confirmed that TALL-104 cell/glioma cell interactions resulted in tumor cell apoptosis. Cytokines are secreted when TALL-104 cells are coincubated with brain tumor cells; however, morphologic analysis assays revealed that the soluble factors contained within clarified supernates obtained from 4 h coincubates added back to brain tumor cell cultures did not trigger the glioma apoptosis. TALL-104 cells do not express Fas ligand, even upon coincubation with glioma targets, which suggests that the Fas/Fas ligand apoptotic pathway is not likely responsible for the cell injury observed. We obtained evidence that cell injury is calcium dependent and that lytic granule exocytosis is triggered by contact of TALL-104 cells with human glioma cells, suggesting that this pathway mediates glioma cell apoptosis and necrosis.

Human alloreactive CTL interactions with gliomas and with those having upregulated HLA expression from exogenous IFN-gamma or IFN-gamma 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.

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

In 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 continuous in vitro exposure to gamma-interferon (gammaIFN) 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 by in situ transduction of the tumor with retroviral vectors coding for gammaIFN, 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 murine gammaIFN gene. By limiting dilution, clones of these cells, designated 9Lgamma 7, 9Lgamma tk8, and 9Lgamma tk10, which produced similar levels of gammaIFN (383-411 ng gammaIFN/10(6) cells/24 h) were isolated. The production of gammaIFN by one clone, 9Lgamma 7, was stable when monitored over 6 weeks in 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, 9Lgamma 7 had approximate 6- and 1.5-fold increases in the relative antigen densities of MHC I and II, respectively. Addition of exogenous gammaIFN to 9Lgamma 7 cultures did not significantly increase the MHC expression. In cytotoxicity assays, 9Lgamma 7 cells, or 9Lgamma 7 incubated with exogenous gammaIFN, were better targets of aCTL than the parental 9L cells. The growth rate of 9Lgamma-transduced cells was decreased compared to the wildtype cells both in vitro and in vivo. Proliferation studies with transwell plated 9L, 9Lgamma 7, and 9Lgamma tk10 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 9Lgamma 7 tumors in rat brains given identical inoculums similarly demonstrated significantly reduced 9Lgamma 7 tumor volumes at various timepoints, indicative of slower growth of the gammaIFN-producing tumors.

Effects of IFN-gamma and interleukin-1beta 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-gamma (IFN-gamma) or interleukin-1beta (IL-1beta) 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-gamma (0-2000 U/ml) or recombinant human IL-1 (rHUIL-1) (0-1000 U/ml) or both. By 48 h, IFN-gamma (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-gamma did not further affect the induction of class I on 9L cells more than that achieved with IFN-gamma alone. 9L cells with upregulated MHC class I and ICAM-1 expression were more sensitive to lysis by aCTL in in vitro cytotoxicity assays, regardless of whether the precursor aCTL came from naive or from 9L-immunized rats. Furthermore, inhibition of 9L cytotoxicity in assays that included blocking antibodies to MHC class I or to ICAM-1 revealed that T cell receptor (TCR) interactions with MHC class I and that ICAM-1 interactions with lymphocyte function-associated-1 (LFA-1) antigen account for a portion of the glioma lysis by aCTL.