Cytokine induction of VCAM-1 but not IL13Rα2 on glioma cells: a tale of two antibodies

Abstract

The interleukin-13 receptor alpha2 (IL13Rα2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13Rα2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13Rα2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13Rα2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13Rα2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13Rα2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.

Figure 1. Differential recognition of constitutively-expressed versus cytokine-induced IL13Rα2 by commercially available anti-IL13Rα2 antibodies.

Flow cytometric analysis of monocytic line THP-1 and various glioma lines with (A) B-D13-PE (Cell Sciences) or (B) AF146 (R&D Systems) reagents for media alone (black histogram) and cytokine (TNF/IL-4 or TNF/IL-13 overnight; red histogram) conditions. Isotype (iso-PE) and mouse anti-goat-FITC controls are shown as grey histograms. Percent positive and relative fluorescent index (RFI) of MFI cytokine/MFI media is reported for each histogram. (C) Flow cytometric detection of IL13Rα2 for D283 cells engineered to express IL13Rα2 (D283-IL13Rα2; blue histogram) and D283 parental (black histogram) stained with AF146 or B-D13-PE antibodies. All data are representative of more than three experiments each.

Figure 2. Cytokine induction of B-D13 antigen is dependent on mRNA transcription and translation.

(A) IL13Rα2 mRNA levels quantified by qPCR for U251T, PBT003-4, PBT008, and THP-1 cells after overnight culture in media, TNF/IL-4, or TNF/IL-13. mRNA levels were normalized to housekeeping genes (ACTB, UBC, GAPDH and RPLP0). (B, C) B-D13-PE immunoreactivity of PBT008 and THP-1 cells treated with increasing concentrations of (B) transcription blocker Actinomycin D (ActD) (0, 0.06, 0.1, 0.3 µg/ml) or (C) translation blocker Cycloheximide (CHX) (0, 2.5, 5, 10 µg/ml), then either cultured in media alone (black histograms) or stimulated with TNF/IL-4 (red histograms) for 5 hours. All data are representative of 2 separate experiments.

Figure 3. The cytokine-induced cell surface antigen recognized by B-D13-PE does not bind IL-13.

(A) D283 cells engineered to express IL13Rα2 (D283- IL13Rα2) (blue histograms) and D283 parental (black histograms) cells were evaluated by flow cytometry for expression of constitutive IL13Rα2 using the IL13Rα2-specific antibody AF146, and biotinylated recombinant human IL-13 (IL13-bio) followed by PE-conjugated strepavidin (SA-PE). Data are representative of 2 separate experiments. (B) U251T (grown in the absence of cytokines) were evaluated by flow cytometry for constitutive IL13Rα2 expression using AF146, and for binding to IL13-bio/SA-PE in the presence and absence of 10-fold molar excess of recombinant human IL-4 or IL-13. Black histograms represent staining with istoype control antibody or SA-PE alone. Data are representative of 2 separate experiments. (C) THP-1 and PBT008 grown in media alone (black histograms) or induced overnight with TNF and IL-4 (red histograms) were analyzed by flow cytometry for expression of constitutive IL13Rα2 (AF146), for expression of the induced antigen (B-D13-PE), and for binding to IL13-bio/SA-PE. Data are representative of 3 separate experiments. (D) IL13-zetakine⁺ CD8⁺ CTL recognize and kill U251T glioma targets expressing constitutive IL13Rα2 (AF146-positive), but not cytokine-induced PBT003 cells (B-D13-positive). Percentage specific lysis (mean ± S.D.) of triplicate wells is depicted. *, p≤0.0002 using an unpaired Student's t-test to compare U251T vs. PBT003-4 targets. #, p>0.05 using an unpaired Student's t-test to compare PBT003-4 targets with and without overnight cytokine stimulation. Data are representative of at least 2 separate experiments.

Figure 4. The B-D13 antibody recognizes VCAM-1, not IL13Rα2.

(A) Silver stain gel of the second elution (E2) from the B-D13 immunoprecipitation of PBT008 stimulated overnight with TNF. (B) Western blot detecting B-D13 pull-down of VCAM-1 in immunoprecipitation eluates (E1, E2, E3) for PBT008 cells stimulated overnight with TNF. VCAM-1 was not immunoprecipitated in the absence of B-D13 antibody (no antibody: E1, E2, E3). Titrated soluble recombinant human VCAM-1-Fc shows specificity of the VCAM-1 antibody. (C) Western blot detecting B-D13 pull-down of VCAM-1 in immunoprecipitation eluates (E1, E2, E3) for THP-1 cells in both media and cytokine overnight-stimulated conditions. All data are representative of 2 separate experiments.

Figure 5. Antibody immunoreactivity, and soluble ligand competition confirms B-D13-PE specificity for VCAM-1.

(A) Flow cytometric analysis of monocytic line THP-1 and various glioma lines with B-D13-PE (top), or PE-conjugated anti-VCAM-1 (VCAM-1-PE, bottom), after overnight culture in media alone (black histogram) versus cytokine (TNF/IL-4; red histogram) conditions. Data are representative of at least 2 separate experiments. (B) PBT008 cells cultured overnight in media alone (black histogram) versus cytokine (TNF/IL-4; red histogram) conditions were stained with B-D13-PE antibody that was pre-incubated with soluble recombinant human IL13Rα-Fc or VCAM-1-Fc. As a control, cells were stained with B-D13-PE alone. Data are representative of 2 separate experiments.

Figure 6. Evaluation of additional commercially available B-D13 antibodies.

(A) PBT008 cells that had been cultured overnight in media alone (black histogram) versus cytokine (TNF; red histogram) conditions, (B) Parental 293T cells or 293T cells engineered to express either VCAM-1 or IL13Rα2, and (C) U251T cells were stained with VCAM-1-PE, AF146 or various B-D13 reagents – two lots of PE-conjugated B-D13 antibody (B-D13-PE; Cell Sciences) and two to three unconjugated B-D13 antibodies (B-D13-unc) purchased from either Cell Sciences (Cell Sci), Abcam or Santa Cruz as indicated. (B, C) Black histograms represent staining with istoype control antibody or SA-PE alone.

Figure 7. B-D13 reagent appears to contain two distinct monoclonal antibodies.

Soluble receptor competition study evaluating the specificity of IL13Rα2 and VCAM-1 recognition by the B-D13-unc antibodies (Cell Sciences and Santa Cruz) using (A) PBT008 cells cultured overnight in media alone (black histogram) versus cytokine (TNF; blue histogram); or (B) IL13Rα2-expressing U251T cells (blue histogram). Cells were stained with the indicated antibody that was pre-incubated with soluble recombinant human IL13Rα-Fc (purple histograms) or VCAM-1-Fc (green histograms). Relative fluorescence index (RFI) compared to staining without the soluble competitors (i.e., the control/blue histograms) are indicated in each histogram. (C) Unconjugated B-D13 antibodies from Cell Sciences (top) and Abcam (bottom) were reduced and analyzed by LC/MS. Shown is the spectra of the deconvoluted protein masses depicting two distinct mass species for both the heavy and light chains. (D) Extracted ion chromatograms (EIC) for the two light chain species, of the Cell Sciences B-D13-unc reagent from (C).