![Figure 1. CLL cells cocultured with CD154-expressing supportive cells are induced to proliferate. (A-B) CLL cells were labeled with CFSE and cocultured on HeLaCD154 in the presence of IL-4 and IL-10, as described in “Methods.” Cells were collected on days 2, 3, 6, and 10 for analysis by flow cytometry. The results of assays on 2 representative CLL samples are shown in (A) and the fraction of dividing cells observed in 6 patient samples tested is presented in (B). One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used to determine statistically significant differences from day 2. ***P < .001 (mean ± standard error of the mean [SEM]; n = 6). (C-D) CLL cells were labeled with CFSE and cocultured on FibroblastsCD154 in the presence of IL-4 and IL-10, as described in “Methods.” Cells were collected on days 1, 6, and 9 for analysis by flow cytometry. One representative CLL sample out of 4 is shown in (C) and the fraction of dividing cells observed in all 4 patients tested is presented in (D). One-way ANOVA and Tukey’s multiple comparison test were used to determine statistically significant differences from day 1. ***P < .001 (mean ± SEM; n = 4). (E-F) CLL cells were cocultured on FibroblastsCD154 in the presence of IL-4 and IL-10, and at the indicated time, subjected to cell-cycle analysis following PI staining. One representative CLL sample out of 4 is shown in (E) and the fraction of cells in each phase for all 4 patients tested is presented in (F). **P < .01; ***P < .001 (Student t test, mean ± SEM; n = 4). (G-H) CLL cells were cocultured on FibroblastsCD154 in the presence of IL-4 and IL-10, and at the indicated time, subjected to 5-ethynyl-2′-deoxyuridine (EdU) incorporation for a period of 4 hours, as described in “Methods.” One representative CLL sample out of 4 is shown in (G) and the fraction of EdU+ cells observed in all 4 patients tested is presented in (H). **P < .01 (Student t test, mean ± SEM; n = 4). (I) CLL cells from 3 different patients were cocultured on HeLa CD154 or nontransfected HeLa cells in the presence of IL-4 and IL-10. At the indicated days, live CLL-cell counts were assessed by flow cytometry, as described in “Methods,” and are presented as expansion folds relative to day 1. *P < .05 (Student t test, mean ± SEM; n = 3).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/10/10.1182_blood-2014-03-559591/4/1637f1.jpeg?Expires=1735519008&Signature=3BBnFwG-zSIFHh02-x0J66Gynwg3GyX7U1n8YoJrZhABEihulpYzEJYiKFwAPU0CMGIyNTGB80q-2iLL56yZZ3ObFds1uQmp1SiFOJPVv6ebGcb7FF8x5~OlQ0vNU8gBrSbSxyqaNhE4IeTa96Fud15xh6QOQazEs6FqLK87FLZKThqRJ~lQm0VE52PBRQ-WMLvQ5fsg8qIbQmhwyNLeGJZ7TVA3s7WfPSDaTsx1fpQ6W52LXLAiTaYwwtyDU2r~GfF7qIBrDkeUnTdiOBl~kPOBE8Htgwcf8a~uotqEdYOijk8qRUL9014wP3JsmlrgVUwyhJ3Q3yaX06Mb6H0DHg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CLL cells cocultured with CD154-expressing supportive cells are induced to proliferate. (A-B) CLL cells were labeled with CFSE and cocultured on HeLaCD154 in the presence of IL-4 and IL-10, as described in “Methods.” Cells were collected on days 2, 3, 6, and 10 for analysis by flow cytometry. The results of assays on 2 representative CLL samples are shown in (A) and the fraction of dividing cells observed in 6 patient samples tested is presented in (B). One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used to determine statistically significant differences from day 2. ***P < .001 (mean ± standard error of the mean [SEM]; n = 6). (C-D) CLL cells were labeled with CFSE and cocultured on FibroblastsCD154 in the presence of IL-4 and IL-10, as described in “Methods.” Cells were collected on days 1, 6, and 9 for analysis by flow cytometry. One representative CLL sample out of 4 is shown in (C) and the fraction of dividing cells observed in all 4 patients tested is presented in (D). One-way ANOVA and Tukey’s multiple comparison test were used to determine statistically significant differences from day 1. ***P < .001 (mean ± SEM; n = 4). (E-F) CLL cells were cocultured on FibroblastsCD154 in the presence of IL-4 and IL-10, and at the indicated time, subjected to cell-cycle analysis following PI staining. One representative CLL sample out of 4 is shown in (E) and the fraction of cells in each phase for all 4 patients tested is presented in (F). **P < .01; ***P < .001 (Student t test, mean ± SEM; n = 4). (G-H) CLL cells were cocultured on FibroblastsCD154 in the presence of IL-4 and IL-10, and at the indicated time, subjected to 5-ethynyl-2′-deoxyuridine (EdU) incorporation for a period of 4 hours, as described in “Methods.” One representative CLL sample out of 4 is shown in (G) and the fraction of EdU+ cells observed in all 4 patients tested is presented in (H). **P < .01 (Student t test, mean ± SEM; n = 4). (I) CLL cells from 3 different patients were cocultured on HeLa CD154 or nontransfected HeLa cells in the presence of IL-4 and IL-10. At the indicated days, live CLL-cell counts were assessed by flow cytometry, as described in “Methods,” and are presented as expansion folds relative to day 1. *P < .05 (Student t test, mean ± SEM; n = 3).
