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Human Autologous In Vitro Models of Glioma Immunogene Therapy Using B7-2, GM-CSF, and IL12

  • Ian F. Parney (a1), Maxine A. Farr-Jones (a1), Kevin Kane (a2), Lung-Ji Chang (a2) and Kenneth C. Petruk (a1)...

Abstract

Background:

Cancer immunogene therapy is based on vaccination with radiated, autologous tumor cells transduced with immunostimulatory genes. To help determine an optimal glioma immunogene therapy strategy, we stimulated lymphocytes with autologous human glioma cells transduced with B7-2 (CD86), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or interleukin-12 (IL12).

Methods:

A human glioma-derived cell culture (Ed147.BT) was transduced with B7-2, GM-CSF, and/or IL12 using retroviral vectors. Autologous peripheral blood mononuclear cells (PBMC) were co-cultured with irradiated gene-transduced tumor alone or a combination of radiated wild type and gene-transduced cells. Peripheral blood mononuclear cells proliferation was determined by serial cell counts. Peripheral blood mononuclear cells phenotype was assessed by flow cytometry for CD4, CD8, and CD16. Anti-tumor cytotoxicity was determined by chromium-51 (51Cr) release assay.

Results:

Peripheral blood mononuclear cells cell numbers all decreased during primary stimulation but tumor cells expressing B7-2 or GM-CSF consistently caused secondary proliferation. Tumors expressing B7-2 and GM-CSF or B7-2, GM-CSF, and IL12 consistently increased PBMC CD8+ (cytotoxic T) and CD16+ (natural killer) percentages. Interestingly, anti-tumor cytotoxicity only exceeded that of PBMC stimulated with wild type tumor alone when peripheral blood mononuclear cells were stimulated with both wild type tumor and B7-2/GM-CSF- (but not IL12) transduced cells.

Conclusion:

PBMC proliferation and phenotype is altered as expected by exposure to immunostimulatory gene-transduced tumor. However, transduced tumor cells alone do not stimulate greater anti-tumor cytotoxicity than wild type tumor. Only B7-2/GM-CSF-transduced cells combined with wild type produced increased cytotoxicity. This may reflect selection of tumor subclones with limited antigenic spectra during retrovirus-mediated gene transfer.

RÉSUMÉ: Introduction:

La thérapie immunogène dans le traitement du cancer est basée sur la vaccination au moyen de cellules tumorales autologues irradiées et transfectée avec des gènes immunostimulants. Nous avons stimulé des lymphocytes au moyen de cellules de gliome humain autologue transfectées avec B7-2, le facteur de croissance des colonies de granulocytes-macrophages (GM-CSF) et/ou lèinterleukine-12 (IL12) afin de définir une stratégie optimale de thérapie immunogène du gliome.

Méthodes:

Une culture de cellules de gliome humain (Ed147.BT) a été transfectée avec B7-2, GM-CSF et/ou IL12 au moyen de vecteurs rétroviraux. Des monocytes provenant de sang périphérique autologue (MSP) ont été co-cultivés seulement avec des cellules transfectées irradiées ou avec une combinaison de cellules de type sauvage irradiées et de cellules transfectées. La prolifération des monocytes du sang périphérique était déterminée par des décomptes cellulaires sériés. Le phénotype des monocytes du sang périphérique était évalué par cytométrie de flux pour CD4, CD8 et CD16. La cytotoxicité anti-tumorale était déterminée par le test de libération du chrome-51.

Résultats:

Le décompte des monocytes du sang périphérique a diminué pendant la stimulation primaire, mais les cellules tumorales exprimant B7-2 ou GM-CSF ont invariablement causé une prolifération secondaire. Les tumeurs exprimant B7-2 et GM-CSF ou B7-2, GM-CSF et IL12 ont invariablement augmenté le pourcentage des MSP CD8+ (T cytotoxiques) et CD16+ (tueurs naturels). Il est intéressant de noter que la cytotoxicité antitumorale était supérieurs à celle des MSP stimulés seulement par la tumeur de type sauvage quand les MSP étaient stimulés par la tumeur de type sauvage et les cellules transfectées avec B7-2/GM-CSF, mais pas avec IL12.

Conclusions:

Tel que prévu, la prolifération et le phénotype des MSP sont modifiés par l’exposition à des cellules tumorales transfectées avec des gènes immunostimulants. Cependant, les cellules tumorales transfectées seules ne stimulent pas une cytotoxicité antitumorale supérieure à celle de la tumeur de type sauvage. Seulement les cellules transfectées avec B7-2/GM-CSF combinées aux cellules de type sauvage ont provoqué une augmentation de la cytotoxicité, ce qui reflète possiblement une sélection de sous-clones tumoraux possédant un spectre antigénique limité pendant le transfert génique médié par le rétrovirus.

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Human Autologous In Vitro Models of Glioma Immunogene Therapy Using B7-2, GM-CSF, and IL12

  • Ian F. Parney (a1), Maxine A. Farr-Jones (a1), Kevin Kane (a2), Lung-Ji Chang (a2) and Kenneth C. Petruk (a1)...

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