Cellular immunity of patients with malignant glioma: prerequisites for dendritic cell vaccination immunotherapy

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Object

Vaccination therapy that uses dendritic cells (DCs) is a promising immunotherapeutic approach. However, it relies on intact cellular immunity and efficient generation of mature DCs, both of which can be impaired in patients with glioma. Therefore, the immune status and ex vivo generation of DC in such patients were studied.

Methods

The frequencies of white blood cell subsets and monocyte-derived, mature DCs in patients with high-grade gliomas and healthy control volunteers were analyzed using flow cytometry.

In the patients, frequencies of lymphocytes, T cells, and B cells were reduced in comparison with the volunteers in the control group, whereas frequencies of neutrophils and monocytes were increased. There were no differences between the two groups in terms of white blood cell counts or the frequency of NK cells and the major T-cell subsets. The responsiveness of T cells to lectin stimulation was normal. For monocytes, lower frequencies of CD80+ and CD86+ cells but not of CD40+ and HLA-DR+ cells were observed in patients. Ex vivo DC generation in a two-step culture protocol in autologous plasma–supplemented medium or in serum-free medium showed only minor differences in CD80 and HLA-DR expression between the patient and control groups. Frequencies of CD83+, CD1a+, CD14, CD40+, and CD86+ cells were comparable. Overall, the serum-free medium was superior to the plasma-supplemented medium and allowed efficient ex vivo generation of CD83+, CD1a+, and CD14 mature DCs.

Conclusions

Only minor defects in the immune status of patients with glioma were observed, which probably would not hamper immunotherapy. Mature DCs can be generated successfully in normal numbers and with typical immunophenotypes from monocytes of patients with glioma, particularly under serum-free conditions.

Abbreviations used in this paper:BrdU = bromodeoxyuridine; DC = dendritic cell; FITC = fluorescein isothiocyanate; GM-CSF = granulocyte–macrophage colony-stimulating factor; GMP = good manufacturing practice; HLA-DR = human leukocyte antigen, locus DR; IL = interleukin; mAb = monoclonal antibody; NK = natural killer; PBMC = peripheral blood mononuclear cell; PHA = phytohemagglutinin; PHA-M = the mucoprotein form of PHA; SEM = standard error of the mean; TNFα= tumor necrosis factor–α.

Article Information

Address reprint requests to: Rüdiger V. Sorg, Ph.D., Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Medical Center, Moorenstrasse 5, Bldg. 14.80, 40225 Düsseldorf, Germany. email: rsorg@itz.uni-duesseldorf.de.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Scatterplots showing frequencies of white blood cell (A), T-cell (B), and monocyte (C) subpopulations in peripheral blood of patients with glioma and healthy control volunteers. Blood samples of patients (white squares) and control group members (black squares) were analyzed. Expression of the molecules indicated on T cells and monocytes for electronically gated CD3+ and CD14+ cells, respectively, was determined using flow cytometry. Median values are shown as horizontal lines; statistical significance was determined according to the Mann–Whitney U-test. Lym = lymphocytes; mon = monocytes; neu = neutrophils.

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    Bar graph showing PHA-responsiveness of mononuclear cells obtained in patients with glioma and healthy control volunteers. Peripheral blood mononuclear cells of patients (P1–P3, white bars) or controls (C1 and C2, black bars) were stimulated for 3 days with PHA, and BrdU incorporation was determined. Results are shown as background-corrected (nonstimulated PBMC BrdU incorporation) mean values ± standard deviations of quadruplicates. OD = optical density.

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    Graphs showing frequencies and immunophenotypes of monocytes (A) and mature DCs (B and C) obtained in patients with glioma. Expression of CD14 and CD83 on immunomagnetically enriched monocytes (A) and after a 9-day tissue culture in plasma-supplemented X-VIVO 15 medium in the presence of GM-CSF and IL-4 for the first 6 days, followed by an additional 3-day incubation period in the presence of GM-CSF, IL-4, and TNFα (B) was determined by flow cytometry. Expression of CD40, CD80, CD86, and HLA-DR after a 9-day tissue culture was also determined by flow cytometry (C). Quadrants in A and B were set according to isotype controls, which are also shown as gray histograms (C). Frequencies of CD14+ and CD83+ cells are indicated.

  • View in gallery

    Scatterplots comparing DC differentiation of CD14+ monocytes obtained in patients with glioma and healthy control volunteers. Ex vivo generation of DCs from patients with malignant glioma (white squares) and healthy control volunteers (black squares) was performed in plasma-supplemented X-VIVO 15 medium. On Day 9, cells were collected and frequencies of CD14+, CD83+, and CD1a+ cells (A); cellular recovery and output of CD14+ and CD83+ cells (calculated for a cellular input of 106 cells [B]); and frequencies of CD40+, CD80+, CD86+, and HLA-DR+ cells (C) were determined. Median values are shown as horizontal lines; statistical significance was determined according to the Mann–Whitney U-test.

  • View in gallery

    Scatterplots comparing DC differentiation of CD14+ monocytes obtained in patients with glioma and healthy controls. Ex vivo generation of DCs from patients with malignant glioma (white squares) and healthy controls (black squares) was performed in serum-free medium. On Day 9, cells were collected and frequencies of CD14+, CD83+, and CD1a+ cells (A); cellular recovery and output of CD14+ and CD83+ cells (calculated for a cellular input of 106 cells [B]); and frequencies of CD40+, CD80+, CD86+, and HLA-DR+ cells (C) were determined. Median values are shown as horizontal lines; statistical significance was determined according to the Mann–Whitney U-test.

  • View in gallery

    Bar graph comparing ex vivo DC generation in plasma-supplemented (white bars) or serum-free medium (black bars). Dendritic cells were generated from monocytes obtained in patients with glioma and healthy controls in a two-step culture protocol using autologous plasma-supplemented medium or serum-free medium. After 9 days of incubation, expression of CD14, CD83, and CD1a was determined by flow cytometry. Data are shown as the mean frequencies of positive cells (± SEM; at least 25 cultures); statistical significance was determined according to the paired Student t-test.

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