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. 2007 Apr;30(3):261-73.
doi: 10.1097/01.cji.0000211339.81211.25.

Cellular and functional characterization of immunoresistant human glioma cell clones selected with alloreactive cytotoxic T lymphocytes reveals their up-regulated synthesis of biologically active TGF-beta

German G Gomez  1 , Carol A Kruse
Affiliations

Cellular and functional characterization of immunoresistant human glioma cell clones selected with alloreactive cytotoxic T lymphocytes reveals their up-regulated synthesis of biologically active TGF-beta

German G Gomez et al. J Immunother. 2007 Apr.

Abstract

Two immunoresistant (IR) glioma cell variants, 13-06-IR29 and 13-06-IR30, were cloned from 13-06-MG glioma cell populations after receiving continuous immunoselective pressure from multiple alloreactive cytotoxic T lymphocyte (aCTL) preparations. Reapplication of aCTL immunoselective pressure to the IR clones, displaying a partial regain in sensitivity to aCTL after removal of the selective pressure, restored the resistance. The IR variants exhibited cross-resistance to non-human leukocyte antigen (HLA)-restricted effector cells and gamma-irradiation, but not to carmustine. The IR clones were characterized for factors that might contribute to the immunoresistance. The aCTL adhesion to extracellular matrix extracts derived from either the IR clones or the parental cells was similar and not impaired. Furthermore, aCTL binding to parental cells and IR clones was equal. Down-regulation of the cell recognition molecules, class I HLA or intercellular adhesion molecule-1 (ICAM-1), that would inhibit their recognition by aCTL was not observed on the IR clones. The down-regulation of Fas by the IR clones correlated with their resistance to FasL-induced apoptosis. HLA-G or FasL that might provide an immunotolerant environment or provide a means of counterattack to aCTL, respectively, were not associated with the IR phenotype. The aCTL, coincubated with the IR clones and parental cells, displayed up-regulation of multiple secreted cytokines. A significant up-regulation of bioactive transforming growth factor (TGF)-beta was observed in the IR clones compared with the parental cells. These data suggest that increased secretion of bioactive TGF-beta may inhibit aCTL lysis of the IR clones. Disruption of the TGF-beta signaling pathway may circumvent the resistance.

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Figures

FIGURE 1
FIGURE 1
Reapplication of immunoselective pressure restores the IR phenotype to IR cultures displaying a partial reversion to aCTL resistance. The 13-06-IR29 and 13-06-IR30 clones previously cultured in the absence of immunoselective pressure for 40 and 13 cell doublings, respectively, were thawed and given intermittent aCTL selective pressure for 35 days. After removal of the selective pressure, the susceptibility of the IR clones and parental cells to aCTL lysis was determined at 3 E:T ratios in cytotoxicity assays. Lysis of a partially relevant glioma target, DBTRG-05 MG that displayed only 2 class I HLA alleles in common with 13-06-MG cells was also tested. Asterisks indicate a statistically significant (P ≤ 0.05) decrease in aCTL lysis compared with the parental glioma cells as determined by Student’s paired t test.
FIGURE 2
FIGURE 2
Assessments for cross-resistance of the IR clones to non-HLA–restricted, allogeneic LAK and TALL-104 cells, to carmustine, and to γ-irradiation treatments. A, The percentage lysis ± SEM obtained for parental 13-06-MG glioma cells and IR clones to aCTL, to LAK cells (generated from the peripheral blood mononuclear cells of the aCTL blood donor), and to lethally irradiated TALL-104 cell effectors, all at a 12.5:1 E:T ratio. B, Sensitivity of the parental and variant glioma cells to carmustine at 5 drug concentrations, shown as percentages of viability ± SEM relative to untreated control cells at 100%. C, Sensitivity of the parental and variant glioma cells treated with an unfractionated dose of γ-irradiation (20 Gy). The percentages of viability ± SEM of the radiation treated cells (□) for each glioma and for nontreated control cells (■) ± SEM are given.
FIGURE 3
FIGURE 3
aCTL adhesion to glioma-derived ECM proteins and conjugate formation between aCTL and glioma cells. A, The adhesion of aCTL to IR clone-derived ECM proteins are expressed as a percentage ± SEM of the aCTL adhered to parental ECM proteins, as derived from absorbance measurements. B, Confocal micrograph demonstrates conjugates of adherent CFSE-labeled 13-06-IR29 glioma cells with CellTracker Orange-labeled aCTL. Multiple smaller sized aCTL (arrows) are associated with the larger individual 13-06-IR29 cells. Damage to the glioma cells is readily visualized by the membrane blebbing (arrowheads). Bar = 20 μm. C, Percentages of parental glioma cells and IR clones bound to aCTL ± SEM, as determined by confocal microscopic examination of 5 random 20 × fields for each glioma cell type incubated with aCTL for 30 minutes.
FIGURE 4
FIGURE 4
Expression of classic HLA-ABC class I antigen, nonclassic HLA-G, ICAM-1, and Fas and FasL by parental 13-06-MG glioma cells and 13-06-IR29 and 13-06-IR30 IR clones by flow cytometric analyses. The histograms show the nonspecific binding of isotype and secondary antibodies (filled peaks) and the degree of specific staining (unfilled peaks). The percentages of positive cells are indicated and the relative antigen densities can be ascertained by placement and height of the peak on the abscissa.
FIGURE 5
FIGURE 5
Flow cytometric scattergrams are shown for CFSE-labeled tumor cells that were (bottom row) or were not (top row) coincubated with FasL positive G10 cells for 18 hours at a 5:1 E:T ratio. The percentages of cells that were live, apoptotic, or dead (late apoptotic/necrotic) are given. A, Fas positive Jurkat cells, (B) Jurkat+G10 cells, (C) 13-06-MG cells, (D) 13-06-MG+G10 cells, (E) 13-06-IR29 cells, (F) 13-06-IR29+G10 cells, (G) 13-06-IR30 cells, and (H) 13-06-IR30+G10 cells.
FIGURE 6
FIGURE 6
Parental glioma cells and IR clones are sensitive to calcium-dependent lysis by aCTL. Parental 13-06-MG cells and IR clones, 13-06-IR29 and 13-06-IR30, were coincubated with anti-13-06 aCTL at a 12.5:1 E:T ratio for 4 hours. In some wells the calcium-chelator, EGTA, was added to inhibit perforin and granzyme-mediated cell lysis. The percentages of glioma cells lysed ± SEM, obtained in the presence or absence of EGTA, are shown.
FIGURE 7
FIGURE 7
Detection of TGF-β isoforms secreted by monolayers of the parental glioma cells or IR clones. The rates of total (latent plus active forms) and active forms of (A) TGF-β1 and (B) TGF-β2 secretions were determined by ELISA. Rates of TGF-β secretion are expressed as pg TGF-β/106 cells/24 h ± SEM. Asterisks indicate statistically significant production (P ≤ 0.02) relative to the parental glioma cells by the nonparametric Kruskal-Wallis test.
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