Figure 1
Figure 1. Natural cytotoxicity of CD3+CD56+ CIK correlates with binding to tumor targets and is LFA-1 dependent. (A) PKH26-labeled CIK and CFSE-labeled BJAB cells were mixed and incubated at 37°C for the indicated times with or without 1μM EDTA and analyzed by flow cytometry. Percentage of binding (values indicated in the top right quadrants) is calculated as the portion of CFSE/PKH-26 double-positive events within the PKH-26–positive events. One representative example is shown. (B) Cytotoxicity of CIK cells against BJAB in the presence (white bars) or absence (gray bars) of 1μM EDTA was evaluated at the indicated E/T ratios. Data were mean ± SD collected from 3 independent experiments and were analyzed by Student t test; *P < .05, **P < .01, and ***P < .005. (C) CIK cells binding to BJAB or KARPAS 422 at different times was analyzed by flow cytometry. Data were obtained from ≥ 3 independent experiments and analyzed by Student t test; *P < .05, **P < .01. (D) Cytotoxicity of CIK cells against BJAB or KARPAS 422 was evaluated by calcein-release assay. Data were mean ± SD obtained from ≥ 3 independent experiments and were analyzed by Student t test; *P < .05. (E) Expression of LFA-1 and their ligands (ICAM-1, -2, and -3) was determined, respectively, on CIK cells and BJAB target by flow cytometry. Background staining with the use of an isotype control Ab is shown in each histogram (gray curve). (F) The functional role of LFA-1 in CIK binding to BJAB (left) and in cytotoxicity (right) was evaluated. For binding assays, PKH-26–labeled CIK cells were exposed to saturating concentrations of blocking Ab anti–LFA-1 or medium alone (CTR) for 30 minutes and then incubated with CFSE-labeled BJAB for 15 minutes. For cytotoxicity assays, CIK cells were preincubated with saturating concentration of anti–LFA-1 or medium alone (CTR) for 15 minutes and then tested in a calcein-release assay against BJAB. Data are the mean percentage of lysis obtained with respect to untreated controls. Data were mean ± SD obtained from ≥ 3 independent experiments and were analyzed by Student t test; *P < .05, **P < .01, and ***P < .005 compared with control in the absence of mAbs.

Natural cytotoxicity of CD3+CD56+ CIK correlates with binding to tumor targets and is LFA-1 dependent. (A) PKH26-labeled CIK and CFSE-labeled BJAB cells were mixed and incubated at 37°C for the indicated times with or without 1μM EDTA and analyzed by flow cytometry. Percentage of binding (values indicated in the top right quadrants) is calculated as the portion of CFSE/PKH-26 double-positive events within the PKH-26–positive events. One representative example is shown. (B) Cytotoxicity of CIK cells against BJAB in the presence (white bars) or absence (gray bars) of 1μM EDTA was evaluated at the indicated E/T ratios. Data were mean ± SD collected from 3 independent experiments and were analyzed by Student t test; *P < .05, **P < .01, and ***P < .005. (C) CIK cells binding to BJAB or KARPAS 422 at different times was analyzed by flow cytometry. Data were obtained from ≥ 3 independent experiments and analyzed by Student t test; *P < .05, **P < .01. (D) Cytotoxicity of CIK cells against BJAB or KARPAS 422 was evaluated by calcein-release assay. Data were mean ± SD obtained from ≥ 3 independent experiments and were analyzed by Student t test; *P < .05. (E) Expression of LFA-1 and their ligands (ICAM-1, -2, and -3) was determined, respectively, on CIK cells and BJAB target by flow cytometry. Background staining with the use of an isotype control Ab is shown in each histogram (gray curve). (F) The functional role of LFA-1 in CIK binding to BJAB (left) and in cytotoxicity (right) was evaluated. For binding assays, PKH-26–labeled CIK cells were exposed to saturating concentrations of blocking Ab anti–LFA-1 or medium alone (CTR) for 30 minutes and then incubated with CFSE-labeled BJAB for 15 minutes. For cytotoxicity assays, CIK cells were preincubated with saturating concentration of anti–LFA-1 or medium alone (CTR) for 15 minutes and then tested in a calcein-release assay against BJAB. Data are the mean percentage of lysis obtained with respect to untreated controls. Data were mean ± SD obtained from ≥ 3 independent experiments and were analyzed by Student t test; *P < .05, **P < .01, and ***P < .005 compared with control in the absence of mAbs.

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