Inhibition of CD24 and CD52 signaling rescues T-cell function in CLL. (A-B) PBMCs from HDs and patients with CLL were thawed and preincubated for 1 hour with 1 μg/mL CD24 and 1 μg/mL CD52 antibodies (A) or alemtuzumab (1 μg/mL) (B) and subsequently stimulated with soluble stimulatory CD3/28 antibodies for 2 days. T cells were analyzed for expression of CD25 afterwards (n = 6-9). (C-E) CLL-derived T cells were stimulated with soluble CD3/28 antibodies in the presence or absence of CD24 and CD52 blocking antibodies. Five minutes after activation, the T cells were stained and analyzed for phosphorylation of CD3ζ (C; n = 4), ERK (D; n = 4), and ZAP70 (E; n = 4). (F-G) PBMCs from patients with CLL were cultured for 16 days in the presence of a single dose of CD3/28 antibodies with or without CD24 and CD52 blocking antibodies. During the culturing period, T-cell activation (CD25) and the expression of PD-1 was measured on CD4 (F) and CD8 (G) T cells. (H) 19BBζ CAR T cells were generated from T cells derived from patients with CLL and cocultured in a 1:1 ratio with either JeKo-1 cells or primary CLL cells, a single dose of blocking antibodies for CD24 and CD52 was added to the culture, and, after 1 day, the cell death of JeKo-1 cells or CLL cells was analyzed and specific lysis was calculated. P values were calculated using a t test for panels A,C,E, a paired t test for panel H, or a 1-way ANOVA for panel B. Data are presented as mean ± SEM; ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001. MFI, mean fluorescence intensity.