Treatment of intracranial gliomas with bone marrow—derived dendritic cells pulsed with tumor antigens

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Object. An approach toward the treatment of intracranial gliomas was developed in a rat experimental model. The authors investigated the ability of “professional” antigen-presenting cells (dendritic cells) to enhance host antitumor immune responses when injected as a vaccine into tumor-bearing animals.

Methods. Dendritic cells, the most potent antigen-presenting cells in the body, were isolated from rat bone marrow precursors stimulated in vitro with granulocyte—macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Cultured cell populations were confirmed to be functional antigen-presenting cells on the basis of expressed major histocompatibility molecules, as analyzed by fluorescence-activated cell sorter cytofluorography. These dendritic cells were then pulsed (cocultured) ex vivo with acid-eluted tumor antigens from 9L glioma cells. Thirty-eight adult female Fischer 344 rats harboring 7-day-old intracranial 9L tumors were treated with three weekly subcutaneous injections of either control media (10 animals), unpulsed dendritic cells (six animals), dendritic cells pulsed with peptides extracted from normal rat astrocytes (10 animals), or 9L tumor antigen—pulsed dendritic cells (12 animals). The animals were followed for survival. At necropsy, the rat brains were removed and examined histologically, and spleens were harvested for cell-mediated cytotoxicity assays.

The results indicate that tumor peptide-pulsed dendritic cell therapy led to prolonged survival in rats with established intracranial 9L tumors implanted 7 days prior to the initiation of vaccine therapy in vivo. Immunohistochemical analyses were used to document a significantly increased perilesional and intratumoral infiltration of CD8+ and CD4+ T cells in the groups treated with tumor antigen—pulsed dendritic cells compared with the control groups. In addition, the results of in vitro cytotoxicity assays suggest that vaccination with these peptide-pulsed dendritic cells can induce specific cytotoxic T lymphocytes against 9L tumor cells.

Conclusions. Based on these results, dendritic antigen-presenting cells pulsed with acid-eluted peptides derived from autologous tumors represent a promising approach to the immunotherapy of established intracranial gliomas, which may serve as a basis for designing clinical trials in patients with brain tumors.

Article Information

Address reprint requests to: Linda M. Liau, M.D., Division of Neurosurgery, University of California at Los Angeles School of Medicine, NPI 18-228, 10833 Le Conte Avenue, Los Angeles, California 90095. email: liau@ucla.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Schematic diagram of experimental treatment in which acid-eluted tumor peptide—pulsed dendritic cells are used as a vaccine for brain tumor immunotherapy in a rat model.

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    Photomicrographs of cytospin preparations of dendritic cell—enriched bone marrow cultures. A: Low-power view of adherent clusters of immature precursor cells, which were easily identified by Day 3. Original magnification × 40. B: High-power view of more mature dendritic cells with typical irregular nuclei and short cytoplasmic dendrites (arrow) at Day 5. Original magnification × 100. C: Low-power view of population of fully mature dendritic cells with elongated cytoplasmic veils and stellate processes. Original magnification × 40. D: High-power view of individual mature dendritic cells with typical cloverleaf-shaped nucleus (arrow) and long cytoplasmic dendrites (arrowhead). Original magnification × 400.

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    Flow cytometric analysis of Fc+ and plastic-adherent dendritic cell—enriched bone marrow cultures at Days 4 and 8. A: Scatterplot showing dendritic cells gated on the basis of forward- and side-scatter profiles of culture cells. B and C: Graphs showing that 45% of cells within the dendritic cell gate were positive for a rat MHC Class II surface marker (OX6+) at Day 4 (B), whereas 94% of cells within the dendritic cell gate were positive at Day 8 (C). The x and y axes represent log fluorescence intensity and relative cell number, respectively.

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    Kaplan—Meier graph showing data on the effect of immunization with antigen-pulsed dendritic cells (DC) on survival of rats with 9L intracranial tumors. Treatment with dendritic cells pulsed with acid-eluted 9L peptides resulted in 58% of animals surviving at 31 days compared with 0% of those given normal brain peptide—pulsed dendritic cells, 0% of animals immunized with unpulsed dendritic cells, and 0% of controls. The median days of survival, range, and probability values for each group are as follows: RPMI-1640 medium: 16 days, range 14 to 24 days, not significant; unpulsed dendritic cells: 17 days, range 15 to 29 days, not significant; dendritic cell pulsed with acid-eluted peptides from normal rat astrocyte cultures: 22 days, range 14 to 31 days, not significant; dendritic cell pulsed with acid-eluted 9L tumor peptides: 35 days, range 14 to more than 80 days, p = 0.027.

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    Photomicrographs showing representative hematoxylin and eosin—stained sections from tumor-bearing rats treated with 9L peptide—pulsed dendritic cells. A–C: Brain sections from an experimental animal that died early (21 days after tumor implantation). Low-power view (A) shows a large intracranial tumor mass. Higher-power views (B and C) of the boundary between normal brain tissue and tumor. Note the lymphocytic accumulation within the tumor but no significant inflammation outside the immediate peritumoral area. The normal brain tissue is marked by an asterisk, and the tumor is marked by a T. D and E: Brain sections from an experimental animal that was a long-term survivor (> 60 days postimplant). Low-power view (D) shows absence of tumor at the implantation site. Higher-power view (E) shows some small areas of necrosis but no appreciable inflammatory infiltrate within the brain parenchyma. Original magnifications × 20 (A); × 200 (B); × 400 (C); × 20 (D); × 200 (E).

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    Photomicrographs showing immunohistochemical staining of brain slices from tumor-bearing rats treated with control media (A) and 9L antigen—pulsed dendritic cells (B and C). The brown staining (CD8+ T cells) is markedly more infiltrative within the tumor treated with antigen-pulsed dendritic cells (B) compared with controls (A), which indicates increased intratumoral T-cell activation. High-power magnification (C) of the area of brown staining (boxed area in B) shows cells resembling tumor-infiltrating lymphocytes (white arrows). Thionine, original magnifications × 100 (A and B); × 400 (C).

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    Graphs showing induction of specific CTL lytic activity against 9L tumor cells in vitro by immunization with dendritic cells (DC) pulsed with acid-eluted tumor peptides. Tumor-bearing Fischer 344 rats were immunized three times with either dendritic cells pulsed with acid-eluted 9L peptides or control media. At necropsy, spleens were harvested and splenocytes were restimulated with irradiated 9L cells in vitro. Cytotoxic activity was then measured using a europium release assay as described in Materials and Methods. This experiment was repeated five times, with splenocytes from two to three rats per assay. The results of each of the five assays are shown in separate graphs. Error bars indicate 1 standard deviation of the mean. Statistical significance between the 9L peptide—pulsed dendritic cell—treated and control media—treated groups based on paired Student's t-tests are as follows: p = 0.39 (not significant [A]); p = 0.50 (not significant [B]); p = 0.037 (C); p = 0.006 (D); p < 0.001 (E).

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