Article Figures & Data
Figures
- Fig. 1.
Functionality of HER2-specific T cells from subjects with GBM. HER2-specific and nontransduced (NT) T cells, generated from newly diagnosed GBM patients, were cocultured with HER2-positive (U373 or Daoy) or HER2-negative (MDA-MB-468) cells. A, only HER2-specific T cells killed HER2-positive targets in a 4-h 51Cr release cytotoxicity assay; nontransduced T cells did not. The HER2-negative cell line MDA-MB-468 was not killed by HER2-specific or nontransduced T cells. Points, mean of all patients; bars, SD. Results from experiments done for nine GBM patients in triplicates are shown. B, in coculture assays done at a 1:1 T-cell to tumor cell ratio, the IFN-γ and IL-2 concentration was determined in the coculture supernatant 24 to 48 h after stimulation. Only HER2-specific T cells produced IFN-γ and IL-2 after exposure to HER2-positive cells in comparison with nontransduced T cells. Results from experiments done in duplicates are shown.
- Fig. 2.
HER2 protein expression on primary GBM. A, FACS analysis. Primary GBM cells from freshly excised tumors in short-term culture were stained for HER2 expression. Open curves, isotype control; solid curves, HER2. Nine of 10 tumor cell lines expressed HER2 on the cell surface. B, using the HER2-specific mouse monoclonal antibody NCL-L-CB11 (Novocastra), HER2 expression was confirmed on the corresponding paraffin-embedded sections. One tumor had no detectable HER2 protein expression using both methods (patient 8). Magnification, ×200.
- Fig. 3.
HER2-specific T cells kill autologous HER2-positive GBM and are activated in coculture. A, the cytolytic activity of T cells expressing HER2 CAR was determined in a standard 4-h chromium release assay. There was always an increase of cytolytic activity of HER2-specific T cells above background (nontransduced T cells) against autologous HER2-positive GBMs. As controls, the HER2-positive GBM cell line U373 and the HER2-negative MDA-MB-468 were used. HER2-specific T cells from all patients killed U373 cells, whereas MDA-MB-468 was not killed (shown for patient 4). B, HER2-specific T cells (black columns) or nontransduced T cells (white columns) from GBM patients were cocultured with autologous tumor cells or HER2-negative control cells (MDA-MB-468; hatched columns) in a 1:1 ratio. Twenty-four to 48 h after stimulation, the cytokine concentration in the medium was determined by ELISA. HER2-specific T cells produced IFN-γ and IL-2 after stimulation with eight of nine HER2-positive tumor samples. No cytokine release was seen with nontransduced T cells. Median cytokine levels for all patients are shown for U373 (HER2-positive control) and MDA-MB-468 (HER2-negative control). Results of experiments done in duplicates are shown.
- Fig. 4.
HER2-specific T cells target primary GBM stem cells. A, primary GBM cells from three patients (GBM patients 2, 3, and 5) were stained for CD133 and isolated using high-speed sorting. Approximately 3% to 5% of the total primary GBM cell population was CD133 positive. B, this CD133-positive cell compartment was uniformly HER2 positive. Moreover, in all three tumors analyzed, the CD133-positive GBM stem cells expressed higher levels of HER2 in comparison with the CD133-negative tumor cell population. C, in a 4-h 51Cr release assay, HER2-specific T cells from these three patients killed autologous CD133-positive cells as well as their CD133-negative counterparts. Autologous nontransduced T cells induced no appreciable killing.
- Fig. 5.
Adoptively transferred HER2-specific T cells induce regression of autologous GBM xenografts in vivo. Primary GBM cells (5 × 104) from patient 2 (two mice per group), patient 3 (three mice per group), and patient 5 (three mice per group) were injected stereotactically into the caudate nucleus of 9- to 12-wk-old SCID mice followed by intratumoral injection of 2 × 106 autologous HER2-specific or nontransduced T cells 6 d after tumor inoculation. A, tumors grew progressively in untreated mice as shown for two representative animals (top row) and in mice receiving nontransduced T cells (middle row), whereas tumors regressed over a period of 2 to 5 d in response to a single injection of autologous HER2-specific T cells generated from the same patient (bottom row). B, quantitative bioluminescence imaging. Autologous HER2-specific T cells induced tumor regression when compared with nontransduced T cells (two-tailed P = 0.002, Mann-Whitney U test). Solid arrows, time of T-cell injection; open arrows, background luminescence (mean, ∼105 photons/s/cm2/sr); n, number of animals tested in each group. C, Kaplan-Meier survival curve. Survival analysis done 60 d after tumor establishment. Mice treated with autologous HER2-specific T cells had a significantly longer survival probability (P < 0.001) in comparison with untreated mice and mice that received nontransduced T cells. D, 1 × 104 CD133-positive GBM cells from patient 2 were injected as described above followed by intratumoral injection of 2 × 106 autologous HER2-specific or nontransduced T cells 8 d after tumor inoculation. Whereas tumors in animals treated with nontransduced T cells (n = 4) continued to grow exponentially, all of the animals treated with autologous HER2 T cells (n = 4) regressed, with two of these animals having no detectable tumors within 6 d after T-cell injection.
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Volume 16, Issue 2
