Hilal Arnouk

Extracellular Targeting of Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 170 Enhances Tumor Immunity to a Poorly Immunogenic Melanoma

We have demonstrated previously that immunization with tumor-derived endoplasmic reticulum (ER) chaperone glucose-regulated protein 170 (grp170) elicits potent antitumor immunity. In the present study, we determine the impact of extracellular targeting grp170 by molecular engineering on tumor immunogenicity and potential use of grp170-secreting tumor cells as a cancer vaccine. grp170 depleted of ER retention sequence “KNDEL,” when secreted by B16 tumor cells, maintained its highly efficient chaperoning activities and was significantly superior to both hsp70 and gp96. The continued secretion of grp170 dramatically reduced the tumorigenicity of B16 tumor cells in vivo, although the modification did not alter its transformation phenotype and cell growth rate. C57BL/6 mice that rejected grp170-secreting B16 tumor cells (B16-sgrp170) developed a strong CTL response recognizing melanocyte differentiation Ag TRP2 and were resistant to subsequent tumor challenge. B16-sgrp170 cells also stimulated the production of proinflammatory cytokines by cocultured dendritic cells. Depletion studies in vivo indicate that NK cells play a primary role in elimination of viable B16-sgrp170 tumor cells inoculated into the animals, whereas both NK cells and CD8+ T cells are required for a long-term protection against wild-type B16 tumor challenge. Both the secreted and endogenous grp170, when purified from the B16 tumor, exhibited potent tumor-protective activities. However, the B16-sgrp170 cell appears to be more effective than tumor-derived grp170. Thus, molecular engineering of tumor cell to release the largest ER chaperone grp170 is capable of eliciting innate as well as adaptive immune responses, which may provide an effective cell-based vaccination approach for cancer immunotherapy.

Cancer immunotherapy and heat-shock proteins: promises and challenges

Recent mechanistic studies on the role of heat-shock proteins (HSPs) to induce innate and adaptive immune responses have resulted in conflicting reports. Whereas some groups reported that HSPs have direct immunological function, others emphasised the endotoxin contamination of HSP preparations and questioned the antigen-specificity of HSP vaccines. The present review will discuss these issues and suggest that HSPs have diverse and distinct immunological functions that could be superimposed on effects resulting from endotoxin contamination or misunderstood by using experimental procedures with inadequate controls. To understand the actual function of HSPs in their interaction with the immune system, methods and procedures need to be optimised and appropriate controls need to be used. These points should also clarify the conflicting findings about HSPs and promote our knowledge about other immuologically important components that may be present in HSP preparations.

Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.

BackgroundInfection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features.ResultsSix replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19–21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27).ConclusionThis large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

Characterization of a Putative Ovarian Oncogene, Elongation Factor 1α, Isolated by Panning a Synthetic Phage Display Single-Chain Variable Fragment Library with Cultured Human Ovarian Cancer Cells

Purpose: In an effort to identify cell surface targets and single short-chain antibody (scFv) for ovarian cancer therapy, we used a phage display approach to isolate an antibody with high reactivity against ovarian cancer. Experimental Design: A phage scFv library was subjected to panning against human SK-OV-3 ovarian cancer cells. A clone with high reactivity was selected and tested in immunoperoxidase staining on a panel of normal tissues and ovarian carcinoma. Using immunoprecipitation, a differentially expressed band was analyzed by mass spectrometry. The antigen subclass was characterized with reverse transcription-PCR on cDNA library of normal tissues, and 91 ovarian cancer specimens, and correlated with clinicohistopathologic characteristics. Results: Ninety-six individual scFv clones were screened in ELISA following panning. scFv F7 revealed high reactivity with ovarian cancer cell lines and showed intense staining of 15 fresh ovarian cancer specimens and no staining of a panel of normal tissues. A 40-kDa protein was identified to be translation elongation factor 1α1 (EEF1A1; P < 0.05). The expression of EEF1A2, a highly homologous and functionally similar oncogene, was found to be restricted only to the normal tissues of the heart, brain, and skeletal muscle. Aberrant EEF1A2 mRNA expression was found in 21 of 91 (23%) of ovarian cancer specimens and significantly correlated with increased likelihood of recurrence (P = 0.021). Conclusions: scFv F7 may represent an ovarian cancer–specific antibody against translation EEF1A family of translational factors. We propose that EEF1A2 may be a useful target for therapy of human ovarian cancer.

CMV-Independent Lysis of Glioblastoma by Ex Vivo Expanded/Activated Vδ1+ γδ T Cells

Vδ2neg γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell - mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM.

Tumour secreted grp170 chaperones full-length protein substrates and induces an adaptive anti-tumour immune response in vivo

Purpose: We employed a grp170-secreting tumour cell system to determine whether tumour cells engineered to secrete grp170 generate an antitumour-specific immune response. Further, we examine the possibility that secreted grp170 can bind to and co-transport out of tumour cells full-length tumour antigens that may play a role in the anti-tumour immune response. Materials and methods: Wild type Colon-26 and Colon-26 engineered to secrete grp170 were subcutaneously inoculated into BALB/c mice. Tumour growth was monitored, and variations in immunoregulatory mechanisms were evaluated using immunohistochemistry, lymphocyte depletion, ELISpot assays, and Western blot analysis. Results: Immunisation of animals with grp170-secreting tumour cells results in rejection of the tumour by induction of antigen-specific, CD8-dependent immune responses. The secreted grp170 is able to deliver full-length tumour antigens to the tumour microenvironment, thus making them available for uptake by antigen presenting cells (APCs) to initiate tumour-specific immune responses. Conclusions: These data parallel our studies showing that hsp110 or grp170 are able to chaperone full-length proteins, and when complexed with protein antigens and used as vaccines, these complexes elicit immune responses in vivo against the protein antigens. This cell-based approach has the potential to be utilised as a tumour-specific vaccine in tumours of various histological origins.

Erratum: Emergence of immune escape variant of mammary tumors that has distinct proteomic profile and a reduced ability to induce "danger signals" (Breast Cancer Research and Treatment DOI: 10.1007/s10549-005- 9044-4)

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