CLL tumor cells induce altered LFA-1 – mediated Rho GTPase activity. (A) The graphs show healthy donor CD3 T cells that were cocultured (48 hours) with either CLL cells (untreated or pretreated with an anti-CD54 neutralizing mAb to block CLL/T cell contact) or third-party healthy B cells (control). T cells from primary coculture or from (B) untreated or lenalidomide (Lenalid.)–treated CLL patients were then purified before exposure to CXCL12 chemokine and LFA-1 cross-linking (mAb 38). T cells were then lysed, and (Bi) RhoA, (Bii) Rac1, and (Biii) Cdc42 activity were measured using G-LISA assays (absorbance at 490 nm). (C) The colored columns show the combined mean activation signal ± SD from 6 CLL patients or healthy donor T cell controls (mean fluorescence expression ± SD). Normalized MFI of Rap1 immunofluorescent staining at the T-cell plasma membrane (C, white box) and (D) total MFI expression of p-MLC from 3 independent experiments (mean ± SD) examining untreated or lenalidomide-treated T cells from CLL patients compared with age-matched healthy donors cells migrating on immobilized CD54 are shown. Representative images of Rap1 and p-MLC staining visualized using confocal microscopy (with Alexa Fluor 488 or 546 secondary antibodies, respectively) for untreated and lenalidomide-treated T cells from CLL patients are shown as indicated. Original magnification ×63.*P < .05; **P < .01. ns, nonsignificant findings.