High CD1d expression on CLL cells and defective iNKT cells correlate with disease progression in patients. (A) Representative flow cytometry detection of CD1d expression on CD19⁺CD5⁺ CLL cells and of iNKT cells in total TCRα/β T cells from 1 patient with stable disease (upper panel) and 1 patient with progressive disease (lower panel). (B) Statistically significant inverse correlation between CD1d expression on CLL cells and autologous iNKT cell frequency (left panel, P = .002551) or iNKT cell number (right panel, P = .005213 Spearman’s correlation test). Scatter plot graphs show single patient value (symbols) with superimposed lines representing the estimated linear regression models (46 patients). (C) Shown are the (i) association of a high CD1d expression on CD19⁺CD5⁺ CLL cells (n = 68 patients) with CLL progression, (ii) the absence of significant difference between CD1d expression on nonmalignant CD19⁺CD5^– cells in patients with stable or progressive disease (same cohorts as in [i]), (iii) the correlation between iNKT cell frequency (n = 50 patients), and (iv) number of patients (n = 46) with CLL progression. Box plots depict first quartile, median, third quartile, outliers (circles), and whiskers proportional to IQR. *P < .05; **P < .005; 1-sided Wilcoxon test. (D) Intracellular staining flow cytometry analysis of IFN-γ production by primary circulating iNKT cells from a representative age-matched healthy donor, a CD1d^neg/low CLL patient, and a CD1d^high CLL patient upon ex vivo activation with anti-CD3/anti-CD28 beads or PMA/ionomycin. The percentage of IFN-γ–producing iNKT and T cells is indicated. (E) Frequency of IFN-γ–producing CD4⁺ (filled circle) and CD4^– (empty circle) iNKT cells from age-matched healthy donors, CD1d^neg/low CLL patients, and CD1d^high CLL patients upon α-CD3/α-CD28 bead activation. Graph symbols show single individual values ± SD. *P < .05 Wilcoxon test. (F) Correlation between CD1d expression on CLL cells and the frequency of IFN-γ–producing iNKT cells and T cells upon α-CD3/α-CD28 bead activation. Scatter plot graphs show single patient (n = 15) values for iNKT and T cells (circles) with superimposed lines representing the estimated linear regression models for iNKT cells (solid) and T cells (dashed). Shown is a significant negative correlation between CD1d expression on CLL cells and frequency of IFN-γ–producing iNKT cells (P = .0096) but not T cells (P = .1136; Spearman’s correlation test).