1. Volume 138, Issue 5, Pages 1931-1942.e2 (May 2010)
Radiofrequency Thermal Ablation for Hepatocellular Carcinoma Stimulates Autologous NK-Cell Response 
Alessandro Zerbini, Massimo Pilli, Diletta Laccabue, Guido Pelosi, Atim Molinari, Elisa Negri, Simona Cerioni, Francesco Fagnoni, Paolo Soliani, Carlo Ferrari, Gabriele Missale 
Gastroenterology 
Volume 138, Issue 5, Pages e2 (May 2010)
DOI: /j.gastro
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2. Figure 1
Significant increase of circulating natural killer (NK) cells after thermal ablation. Frequency of lymphocyte subsets in peripheral blood mononuclear cells (PBMCs) were analyzed in 19 patients undergoing radiofrequency thermal ablation (RFA) the day before, 1 and 4 weeks after treatment (patients 1 to 19, Table 1). (A) NK cells are the lymphocyte subset showing the highest increase after thermal ablation. (Left panel) Relative changes to baseline express mean values with error bars of the percent increases or decreases at week 1 and 4 after treatment. (Right panel) Comparison between absolute numbers of NK cells detected the day before, 1 and 4 weeks after treatment. (B) Representative dot plots of NK cells analysis at different time points are shown. A significant increase of CD56+CD3− cells frequency was observed 1 and 4 weeks after RFA. NK cells were analyzed as subpopulation of CD56Dim and CD56bright cells in 30 patients (patients with phenotypic analysis, Table 1).
Gastroenterology  , e2DOI: ( /j.gastro )
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3. Figure 2
Phenotypic characterization of natural killer (NK) cells. Surface expression of activating and inhibitory receptors was analyzed on CD56+CD3− NK cells. Plots illustrate representative analysis. (A) Frequencies of natural cytotoxicity receptors (NKp30 and NKp46) positive NK cells and mean fluorescence intensity (MFI) of CD16 and NKG2D on NK cells are represented. (B) Frequencies of NKG2A-positive NK cells and NK cells expressing inhibitory killer immunoglobulin-like receptor (KIRs, CD158a, CD158b, and CD158e) are represented. MFI was calculated by subtracting the values obtained with each isotype control tested for each phenotypic marker. Average values and statistical differences (t-test for paired data) are shown in each panel.
Gastroenterology  , e2DOI: ( /j.gastro )
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4. Figure 3
Increase of NKG2D-mediated cytotoxicity 4 weeks after thermal ablation. Natural killer (NK) cytotoxic function was assessed using peripheral blood mononuclear cells (PBMCs) at different effector-target (E:T) cell ratios. (A) Mean value of cytotoxicity detected with PBMCs ex vivo (left) or after overnight stimulation with interleukin (IL)-15 (right). (B) Values of cytotoxicity were normalized according to the frequency of CD3−CD56+ NK cells. Differential increase of cytotoxic function was lost following in vitro exposure to IL-15. (C) NK cells were purified by negative selection from 5 patients in order to confirm the profile of response obtained with PBMCs. (Killing efficiency: normalization of the percent target cell lysis to the NK-cells frequency of each patient at the different time points). t-test was used to compare antibody-dependent cell-mediated cytotoxicity (ADCC) between groups. Means and standard deviations are shown.
Gastroenterology  , e2DOI: ( /j.gastro )
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5. Figure 4
Increase of antibody-dependent cell-mediated cytotoxicity (ADCC) 4 weeks after thermal ablation. Peripheral blood mononuclear cells (PBMCs) derived from 10 patients were cultured at different effector-to-target (E:T) ratios with Her/2neu-positive breast cancer cells (MDA-MB-361) pretreated with anti-Her/2neu (trastuzumab-Herceptin) or control antibody (anti-CD20-Rituximab). (A) Mean percentages of ADCC obtained with PBMCs ex vivo (left) and after normalization according to the frequency of CD3−CD56+ NK cells. (B) NK cells were purified by negative selection from 4 patients in order to confirm responses obtained with PBMCs. (Killing efficiency: normalization of the percent target cell lysis to the NK-cell frequency of each patient at the different time points). t-Test (for paired data) was used to compare ADCC between groups. Means and standard deviations are shown.
Gastroenterology  , e2DOI: ( /j.gastro )
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6. Figure 5
Increase of interferon-γ (IFN-γ) production by CD56Bright natural killer (NK) cells after thermal ablation. Peripheral blood mononuclear cells (PBMCs) were incubated overnight with interleukin (IL)-12 either alone or in combination with IL-18, brefeldin-A was added during the last 3 hours. Cells were then stained with monoclonal antibodies to detect IFN-γ+ NK cells. (A) Percentages of IFN-γ+ CD3−CD56Dim NK cells after IL-12 or IL-12/18 PBMCs stimulation. (B) Percentages of IFN-γ+ CD3−CD56Bright NK cells after IL-12 or IL-12/18 PBMCs stimulation. Each dot represents a patient. Horizontal lines illustrate the median. t-Test (for paired data) was used to compare IFN-γ production between groups.
Gastroenterology  , e2DOI: ( /j.gastro )
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7. Figure 6
Natural killer (NK)-cell functional activation and probability of disease-free survival. Patients were divided in 2 groups according to high and low enhancement of IFN-γ production upon IL-12 stimulation and K562 cytotoxicity 4 weeks after thermal ablation. High and low groups were defined dividing patients according to the median value (ie, 50th percentile) of the enhancement of IFN-γ production (A) or cytotoxicity (B). Kaplan-Meier curves show statistically different probability of disease-free survival over an observation period ranging from 12 to 38 months.
Gastroenterology  , e2DOI: ( /j.gastro )
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8. Supplementary Figure 1
Higher frequency of natural killer (NK) cells in patients with lower tumor burden. Basal frequencies of CD6+CD3− cells (gate on lymphocytes) were significantly higher (t-test) in patients with a smaller hepatocellular carcinoma (HCC) cumulative diameter. Significance was maintained 1 week but not 4 weeks after treatment. Patients groups were defined according to the size of HCC nodules: above and below the median value (24 mm) of the cumulative diameters. Frequency of CD56bright/CD56+CD3− cells showed an opposite behavior. In this case comparison of frequencies was not statistically different at all time points.
Gastroenterology  , e2DOI: ( /j.gastro )
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