Evidence for the involvement of gamma delta T cells in the immune response in Rasmussen encephalitis.

BACKGROUND: Rasmussen encephalitis (RE) is a rare neuroinflammatory disease characterized by intractable seizures and progressive atrophy on one side of the cerebrum. Perivascular cuffing and clusters of T cells in the affected cortical hemisphere are indicative of an active cellular immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) and brain-infiltrating lymphocytes (BILs) were isolated from 20 RE surgery specimens by standard methods, and CD3(+) T cell populations were analyzed by flow cytometry. Gamma delta T cell receptor spectratyping was carried out by nested PCR of reversed transcribed RNA extracted from RE brain tissue, followed by high resolution capillary electrophoresis. A MiSeq DNA sequencing platform was used to sequence the third complementarity determining region (CDR3) of δ1 chains. RESULTS: CD3(+) BILs from all of the RE brain specimens comprised both αß and γδ T cells. The median αß:γδ ratio was 1.9 (range 0.58-5.2) compared with a median ratio of 7.7 (range 2.7-40.8) in peripheral blood from the same patients. The αß T cells isolated from brain tissue were predominantly CD8(+), and the majority of γδ T cells were CD4(-) CD8(-). Staining for the early activation marker CD69 showed that a fraction of the αß and γδ T cells in the BILs were activated (median 42%; range 13-91%, and median 47%; range 14-99%, respectively). Spectratyping T cell receptor (TCR) Vδ1-3 chains from 14 of the RE brain tissue specimens indicated that the γδ T cell repertoire was relatively restricted. Sequencing δ1 chain PCR fragments revealed that the same prevalent CDR3 sequences were found in all of the brain specimens. These CDR3 sequences were also detected in brain tissue from 15 focal cortical dysplasia (FCD) cases. CONCLUSION: Neuroinflammation in RE involves both activated αß and γδ T cells. The presence of γδ T cells with identical TCR δ1 chain CDR3 sequences in all of the brain specimens examined suggests that a non-major histocompatibility complex (MHC)-restricted immune response to the same antigen(s) is involved in the etiology of RE. The presence of the same δ1 clones in CD brain implies the involvement of a common inflammatory pathway in both diseases.

Glioma big potassium channel expression in human cancers and possible T cell epitopes for their immunotherapy.

Big potassium (BK) ion channels have several spliced variants. One spliced variant initially described within human glioma cells is the glioma BK (gBK) channel. This isoform consists of 34 aa inserted into the intracellular region of the basic BK ion channel. PCR primers specific for this inserted region confirmed that human glioma cell lines and freshly resected surgical tissues from glioblastoma multiforme patients strongly expressed gBK mRNA. Normal human brain tissue very weakly expressed this transcript. An Ab specific for this gBK isoform confirmed that human glioma cells displayed this protein in the cell membrane, mitochondria, Golgi, and endoplasmic reticulum. Within the gBK region, two putative epitopes (gBK1 and gBK2) are predicted to bind to the HLA-A*0201 molecule. HLA-A*0201-restricted human CTLs were generated in vitro using gBK peptide-pulsed dendritic cells. Both gBK1 and gBK2 peptide-specific CTLs killed HLA-A2⁺/gBK⁺ gliomas, but they failed to kill non-HLA-A2-expressing but gBK⁺ target cells in cytolytic assays. T2 cells loaded with exogenous gBK peptides, but not with the influenza M1 control peptide, were only killed by their respective CTLs. The gBK-specific CTLs also killed a variety of other HLA-A*0201⁺ cancer cells that possess gBK, as well as HLA-A2⁺ HEK cells transfected with the gBK gene. Of clinical relevance, we found that T cells derived from glioblastoma multiforme patients that were sensitized to the gBK peptide could also kill target cells expressing gBK. This study shows that peptides derived from cancer-associated ion channels maybe useful targets for T cell-mediated immunotherapy.

Differential expression of interferon-γ and chemokine genes distinguishes Rasmussen encephalitis from cortical dysplasia and provides evidence for an early Th1 immune response.

BACKGROUND: Rasmussen encephalitis (RE) is a rare complex inflammatory disease, primarily seen in young children, that is characterized by severe partial seizures and brain atrophy. Surgery is currently the only effective treatment option. To identify genes specifically associated with the immunopathology in RE, RNA transcripts of genes involved in inflammation and autoimmunity were measured in brain tissue from RE surgeries and compared with those in surgical specimens of cortical dysplasia (CD), a major cause of intractable pediatric epilepsy. METHODS: Quantitative polymerase chain reactions measured the relative expression of 84 genes related to inflammation and autoimmunity in 12 RE specimens and in the reference group of 12 CD surgical specimens. Data were analyzed by consensus clustering using the entire dataset, and by pairwise comparison of gene expression levels between the RE and CD cohorts using the Harrell-Davis distribution-free quantile estimator method. RESULTS: Consensus clustering identified six RE cases that were clearly distinguished from the CD cases and from other RE cases. Pairwise comparison showed that seven mRNAs encoding interferon-γ, CCL5, CCL22, CCL23, CXCL9, CXCL10, and Fas ligand were higher in the RE specimens compared with the CD specimens, whereas the mRNA encoding hypoxanthine-guanine phosphoribosyltransferase was reduced. Interferon-γ, CXCL5, CXCL9 and CXCL10 mRNA levels negatively correlated with time from seizure onset to surgery (P <0.05), whereas CCL23 and Fas ligand transcript levels positively correlated with the degree of tissue destruction and inflammation, respectively (P <0.05), as determined from magnetic resonance imaging (MRI) T2 and FLAIR images. Accumulation of CD4+ lymphocytes in leptomeninges and perivascular spaces was a prominent feature in RE specimens resected within a year of seizure onset. CONCLUSIONS: Active disease is characterized by a Th1 immune response that appears to involve both CD8+ and CD4+ T cells. Our findings suggest therapeutic intervention targeting specific chemokine/chemokine receptors may be useful in early stage RE.

Cellular and vaccine therapeutic approaches for gliomas.

Despite new additions to the standard of care therapy for high grade primary malignant brain tumors, the prognosis for patients with this disease is still poor. A small contingent of clinical researchers are focusing their efforts on testing the safety, feasibility and efficacy of experimental active and passive immunotherapy approaches for gliomas and are primarily conducting Phase I and II clinical trials. Few trials have advanced to the Phase III arena. Here we provide an overview of the cellular therapies and vaccine trials currently open for patient accrual obtained from a search of http://www.clinicaltrials.gov. The search was refined with terms that would identify the Phase I, II and III immunotherapy trials open for adult glioma patient accrual in the United States. From the list, those that are currently open for patient accrual are discussed in this review. A variety of adoptive immunotherapy trials using ex vivo activated effector cell preparations, cell-based and non-cell-based vaccines, and several combination passive and active immunotherapy approaches are discussed.

FAPP2 gene downregulation increases tumor cell sensitivity to Fas-induced apoptosis.

The gene for phosphatidylinositol-4-phosphate adaptor-2 (FAPP2) encodes a cytoplasmic lipid transferase with a plekstrin homology domain that has been implicated in vesicle maturation and transport from trans-Golgi to the plasma membrane. The introduction of ribozymes targeting the FAPP2 gene in colon carcinoma cells induced their apoptosis in the presence of Fas agonistic antibody. Furthermore, by quantitative PCR we showed that a siRNA specific to FAPP2, but not a randomized siRNA control, reduced FAPP2 gene expression in tumor cells. Transfection of FAPP2 siRNA into human tumor cells then incubated with FasL resulted in reduction of viable cell numbers. Also, FAPP2 siRNA transfected glioma and breast tumor cells showed significant increases in apoptosis upon incubation with soluble FasL, but the apoptosis did not necessarily correlate with increased Fas expression. These data demonstrate a previously unknown role for FAPP2 in conferring resistance to apoptosis and indicate that FAPP2 may be a target for cancer therapy.

PTEN knockout prostate cancer as a model for experimental immunotherapy.

PURPOSE: Testing immunotherapeutic strategies for prostate cancer has been impeded by the lack of relevant tumor models in immunocompetent animals. This opportunity is now provided by the recent development of prostate specific PTEN knockout mice, which show spontaneous development of true adenocarcinoma arising from prostate epithelium and more faithfully recapitulate the human disease than any previous model. We investigated the feasibility of using tumor cells derived from this model to test tumor vaccination and adoptive immunotherapeutic strategies for prostate cancer. MATERIALS AND METHODS: PTEN-CaP8 adenocarcinoma cells derived from the biallelic PTEN knockout prostate cancer model were used to vaccinate nontumor bearing litter mates. Tumor specific effector cells were generated from splenocytes of vaccinated mice by mixed lymphocyte-tumor reactions, and antiproliferative effects and cytokine generation were examined in vitro. The effect of vaccination or adoptive immunotherapy on luciferase marked PTEN-CaP8 subcutaneous tumors was monitored by tumor volumetric measurements and noninvasive bioluminescence imaging. RESULTS: Vaccination of litter mate mice with irradiated PTEN-CaP8 cells showed a significant prophylactic effect against the subsequent tumor challenge. Effector cells harvested from vaccinated litter mates showed significant interferon-gamma secretion upon co-incubation with PTEN-CaP8 target cells and they were capable of efficient target cell growth inhibition in vitro. Intratumor adoptive transfer of effector cells resulted in significant growth inhibition of preestablished prostate tumors in vivo. CONCLUSIONS: The PTEN knockout model serves as a highly useful model in which to investigate tumor cell vaccination and adoptive immunotherapeutic strategies in the context of true adenocarcinoma of the prostate. This model should accelerate efforts to develop effective immunotherapies for human prostate cancer.

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.

Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity.

BACKGROUND: Prodrug-activator gene therapy with Toca 511, a tumor-selective retroviral replicating vector (RRV) encoding yeast cytosine deaminase, is being evaluated in recurrent high-grade glioma patients. Nonlytic retroviral infection leads to permanent integration of RRV into the cancer cell genome, converting infected cancer cell and progeny into stable vector producer cells, enabling ongoing transduction and viral persistence within tumors. Cytosine deaminase in infected tumor cells converts the antifungal prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil, mediating local tumor destruction without significant systemic adverse effects. METHODS: Here we investigated mechanisms underlying the therapeutic efficacy of this approach in orthotopic brain tumor models, employing both human glioma xenografts in immunodeficient hosts and syngeneic murine gliomas in immunocompetent hosts. RESULTS: In both models, a single injection of replicating vector followed by prodrug administration achieved long-term survival benefit. In the immunodeficient model, tumors recurred repeatedly, but bioluminescence imaging of tumors enabled tailored scheduling of multicycle prodrug administration, continued control of disease burden, and long-term survival. In the immunocompetent model, complete loss of tumor signal was observed after only 1-2 cycles of prodrug, followed by long-term survival without recurrence for >300 days despite discontinuation of prodrug. Long-term survivors rejected challenge with uninfected glioma cells, indicating immunological responses against native tumor antigens, and immune cell depletion showed a critical role for CD4+ T cells. CONCLUSION: These results support dual mechanisms of action contributing to the efficacy of RRV-mediated prodrug-activator gene therapy: long-term tumor control by prodrug conversion-mediated cytoreduction, and induction of antitumor immunity.

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.

Temozolomide induces the expression of the glioma Big Potassium (gBK) ion channel, while inhibiting fascin-1 expression: possible targets for glioma therapy.

OBJECTIVE: Temozolomide (TMZ) improves Glioblastoma Multiforme (GBM) patient survival. The invasive behavior of the glioma cells is the cause of GBM relapse. The glioma BK ion channel (gBK) may provide glioma cells with a mechanism to invade surrounding tissue. gBK contains epitopes that cytolytic T lymphocytes (CTLs) can recognize and kill glioma cells. Fascin-1 is an actin crosslinking molecule that supports microvilli; these membrane protrusions provide a physical defense against CTLs. TMZ was investigated to determine its effect on gBK and fascin-1 expression. RESEARCH DESIGN AND METHODS: Human glioma cells cultured in TMZ were analyzed for their altered mRNA and gBK protein levels by using quantitative real time PCR, immunostaining and cellular functional assays. RESULTS: TMZ slowed glioma cell growth and inhibited their transmigratory properties due to loss of fascin-1. TMZ induced increased gBK and HLA expression and allowed these TMZ-treated cells to become better targets for gBK-specific CTLs. CONCLUSIONS: Besides its traditional chemotherapeutic effect, TMZ can have four other targeted pathways: 1) slowed glioma cell growth; 2) inhibited glioma cell transmigration; 3) increased HLA-A2 and gBK tumor antigen production; 4) increased CTL-mediated cytolysis of the TMZ treated glioma cells due to the loss of their defensive membrane protrusions supported by fascin-1.

Efficacy of systemic adoptive transfer immunotherapy targeting NY-ESO-1 for glioblastoma.

BACKGROUND: Immunotherapy is an ideal treatment modality to specifically target the diffusely infiltrative tumor cells of malignant gliomas while sparing the normal brain parenchyma. However, progress in the development of these therapies for glioblastoma has been slow due to the lack of immunogenic antigen targets that are expressed uniformly and selectively by gliomas. METHODS: We utilized human glioblastoma cell cultures to induce expression of New York-esophageal squamous cell carcinoma (NY-ESO-1) following in vitro treatment with the demethylating agent decitabine. We then investigated the phenotype of lymphocytes specific for NY-ESO-1 using flow cytometry analysis and cytotoxicity against cells treated with decitabine using the xCelligence real-time cytotoxicity assay. Finally, we examined the in vivo application of this immune therapy using an intracranially implanted xenograft model for in situ T cell trafficking, survival, and tissue studies. RESULTS: Our studies showed that treatment of intracranial glioma-bearing mice with decitabine reliably and consistently induced the expression of an immunogenic tumor-rejection antigen, NY-ESO-1, specifically in glioma cells and not in normal brain tissue. The upregulation of NY-ESO-1 by intracranial gliomas was associated with the migration of adoptively transferred NY-ESO-1-specific lymphocytes along white matter tracts to these tumors in the brain. Similarly, NY-ESO-1-specific adoptive T cell therapy demonstrated antitumor activity after decitabine treatment and conferred a highly significant survival benefit to mice bearing established intracranial human glioma xenografts. Transfer of NY-ESO-1-specific T cells systemically was superior to intracranial administration and resulted in significantly extended and long-term survival of animals. CONCLUSION: These results reveal an innovative, clinically feasible strategy for the treatment of glioblastoma.

Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity.

Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses.

Radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive T lymphocytes.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

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.

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.

Purified herpes simplex virus thymidine kinase retroviral particles: III. Characterization of bystander killing mechanisms in transfected tumor cells.

An important consequence of the suicide gene therapeutic paradigm is the phenomenon of bystander cell killing, the death of adjacent tumor cells not transduced with the thymidine kinase (TK) gene from herpes simplex virus (HSV) after treatment with the antiviral drug, ganciclovir (GCV). Evidence from quantitative in vitro assays of glioma cell lines suggest that both murine and human gliomas are similar in expressing high sensitivity to the bystander effect. In five of six glial tumors examined, the presence of only 5% of HSV-TK-expressing transduced cells in the culture resulted in >90% tumor cell death/stasis after addition of GCV. Several lines of evidence support gap junction intercellular communication (GJIC) as important in the bystander effect. In vitro metabolic assays, performed with GCV in the medium, indicated that more tumor burden was reduced when culture conditions supported cell-cell contact of parental and HSV-TK-transduced cells. Additionally, a double dye transfer assay showed that cell communication through the gap junction is greatest for glioma, less for melanoma, and much less for colorectal carcinoma cell lines. In vitro metabolic assays with mixtures of TK+/TK- homologous tumor cells confirmed that glioma cell lines were more susceptible to bystander killing than melanomas. Assays with chimeric tumor mixtures of TK+/TK - cells showed that the level of the bystander killing obtained was characteristic of the TK-bystander cells. The in vitro findings were confirmed in vivo with GCV-treated homologous and chimeric tumors composed of TK+/TK- cells. Day 21 mean tumor volumes (MTVs) indicated the growths obtained were characteristic of the bystander activity reflective of the nontransduced cell population. Furthermore, nontransduced, high-GJIC cells in a chimeric tumor mass appeared to effectively bridge between transduced tumor cells and poorly communicating nontransduced cells. Finally, the importance of a gap junction protein, such as connexin-43, in facilitating the bystander effect was demonstrated with the HT29 low-GJIC cell line. When the TK-nontransduced cell population expressed connexin-43, a better bystander kill was achieved compared to the parental counterpart.

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.

Microglia phagocytose alloreactive CTL-damaged 9L gliosarcoma cells.

Intracranial adoptive transfers of alloreactive cytotoxic T lymphocytes (aCTL) for brain tumor treatment were safe and showed promise in preclinical and early clinical trials. To better understand the endogenous immune responses that may ensue following cellular therapy with aCTL, we examined the ability of microglia to phagocytose aCTL-damaged and undamaged rat 9L gliosarcoma cells in vitro and in vivo. In vitro, 5.5+/-0.9% of microglial cells isolated from adult tumor-bearing rat brains phagocytosed aCTL-damaged 9L cells, whereas microglia did not bind to or ingest undamaged 9L cells. Addition of supernates from either 9L cell cultures or from aCTL+9L co-incubate cell cultures to microglia did not significantly alter their ability to bind to or phagocytose damaged glioma cells even though the latter contained T helper 1 and 2 cytokines. At 3 days following intracranial 9L cell infusion, 17.5+/-0.1% of the microglia phagocytosed CFSE-labeled aCTL-damaged 9L tumor cells within the adult rat brain, confirming the in vitro data. The results suggest that microglia within the tumor microenvironment of the adult rat glioma model selectively remove damaged, but not undamaged, glioma cells.

Ribozyme to proliferating cell nuclear antigen to treat proliferative vitreoretinopathy.

PURPOSE: A DNA-RNA chimeric ribozyme was developed that targets the mRNA of a cell cycle regulatory protein, proliferating cell nuclear antigen (PCNA). The hypothesis was that inhibition of PCNA, essential in DNA replication, would decrease the proliferation of cells that are involved in formation of granuloma after surgical procedures in the eye. The ability of intravitreous injection of this ribozyme to prevent or inhibit development of proliferative vitreoretinopathy (PVR) was tested in a dispase-induced rabbit PVR model. METHODS: Rabbit genomic DNA encoding PCNA was cloned and sequenced. The cleavage of rabbit PCNA by the chimeric ribozyme was tested in vitro. Delivery of the ribozyme to rabbit retinal pigment epithelial (RPE) or fibroblast cells and its effects on proliferation of fibroblasts were examined. The stability of the ribozyme in vitreous fluid and serum was studied as well. In the dispase-induced rabbit model of PVR, the ability of the PCNA ribozyme to prevent or inhibit development of PVR and retinal detachment (RD) was tested. Experimental groups receiving intravitreous PCNA ribozyme, with or without a lipid vehicle, were compared with sham-treated control groups. Progression of PVR in rabbit eyes was followed by indirect ophthalmic examination and observations documented by fundoscopic photography, gross pathology, and histopathology. RESULTS: The chimeric ribozyme targeted a specific sequence in the rabbit PCNA that was identical with that in the human. In vitro cleavage assays confirmed the ability of the ribozyme to cleave the mRNA of PCNA. The catalytic efficiency in vitro, calculated as k(2)/K(m)(app), was 0.26 microM(-1) x min(-1). In vitro studies with fluoresceinated ribozyme indicated that lipid vehicles facilitated delivery of the ribozyme into cells causative of PVR (RPE and fibroblasts); however, the PCNA ribozyme decreased the proliferation of fibroblasts, with or without lipid vehicle. The ribozyme displayed good stability in vitreous fluid, whereas, it degraded quite rapidly in serum. In animal experiments, rabbits in sham-treated groups usually exhibited development of severe PVR characterized by focal traction or RD. Animals in the PCNA ribozyme-treated groups usually did not exhibit an RD. If they did have RD, it was small and localized, or focal tractions developed that did not progress to the degree that the sham-treated animal eyes did over the follow-up period. The in vivo use of a lipid delivery vehicle resulted in a precipitate; however, an effective naked ribozyme dose was identified that did not cause this side effect. CONCLUSIONS: In addition to validating the newly developed dispase PVR rabbit model, the results indicate that ribozyme targeted against the cell cycle agent PCNA is efficacious in the treatment or prevention of PVR in the rabbit eye. These experiments suggest that chimeric ribozyme targeted against PCNA may have a therapeutic or preventative role in humans.