Lack of NKT cells in Tcl1 mice accelerates leukemia onset and progression. (A) Representative flow cytometry analysis of normal (IgM⁺B220^high) and malignant (IgM⁺B220^low) CD19⁺ B cells from the blood of the indicated mouse strains at 30 weeks of age and CD1d expression on healthy and malignant B cells in Tcl1 mice at 15 and 45 weeks of age. (B) Longitudinal analysis of leukemia progression in peripheral blood (n = 16 mice per group). Temporal average kinetic curves describing malignant B-cell frequency as a function of age (50-week range) were calculated for each mouse group (thick colored lines) by mixed-effect model analysis. Age of mice at which CLL frequency reaches 50% of the asymptotic value is 28.5 weeks for Tcl1-CD1d^−/−, 30.6 weeks for Tcl1-Jα18^−/−, and 34.1 weeks for Tcl1 mice. Student t test: Tcl1-CD1d^−/− vs Tcl1-Jα18^−/− mice, P = .0084; Tcl1-Jα18^−/− vs Tcl1 mice, P = .00018. Thin gray curves smoothed via local regression with Gaussian kernel describe the kinetic (30-week) range of malignancy expansion for each animal; the average kinetics (thick colored lines) are superimposed for each strain. (C) CLL disease score of hematoxylin and eosin (H&E)–stained tissue sections from different organs from Tcl1 and Tcl1-Jα18^−/− mice at different ages as indicated, defined by a pathologist in a blinded fashion on the basis of the degree of leukemia infiltration and disruption of the normal organ architecture in spleen (SPL), liver (LV), kidney (KD), lung (LG), lymph node (LN), and bone marrow (BM). Box plots depict first quartile, median, and third quartile. The outlier values (circles) less than interquartile range (IQR) –1.5 or greater than IQR +1.5 are shown. Vertical bars represent whiskers indicating the distance from the smallest (lower bar) and the highest (upper bar) nonoutlier values from the first and third quartile, respectively. *P < .05 Wilcoxon test. (D) H&E staining of tissue sections from spleens of 1 representative Tcl1 (left panel) and Tcl1-Jα18^−/− (right panel) mouse at 17 weeks of age. The splenic architecture of red pulp (RP) in the Tcl1 mouse is maintained, whereas it is substituted by CLL cells in Tcl1-Jα18^−/− mice (original magnification ×40). Images were acquired with Zeiss AxioImager M2m equipped with Nuance FX Multispectral Tissue Imaging System and Nuance Acquisition Software. (E) Tcl1 and wt mice at 20 and 50 weeks of age received α-GalCer–loaded DCs intravenously, and IFN-γ serum level was measured by ELISA at the indicated times. Each curve represents the IFN-γ released by each mouse. One representative result of 3 independent and consistent experiments is shown. **P ≤ .005 Wilcoxon test. (F) Purified CLL from Tcl1 and Tcl1-CD1d^−/− mice and purified B cells from wt mice preloaded or not with α-GalCer were cultured for 48 hours with iNKT cell lines from wt mice with or without anti-CD1d blocking mAb as indicated, and the released IFN-γ was measured by ELISA. Graphs depict mean ± standard deviation (SD). One representative result of 2 independent and consistent experiments is shown. *P < .05; **P ≤ .005; ***P ≤ .0005; ****P ≤ .00005 1-way analysis of variance (ANOVA). (G) In all, 2 × 10⁶ purified and α-GalCer preloaded CLL cells from Tcl1 or Tcl1 CD1d^−/− mice were injected intraperitoneally into C57BL/6N mice (n = 6 per group). The expansion of circulating CLL cells was monitored by flow cytometry at the indicated time points. One of 2 comparable independent experiments is shown. Graphs depict mean values (symbols) ± SD. *P < .05; **P ≤ .005 Wilcoxon test. (H) Posttransplant spleen infiltration by CLL cells. The number of spleen-infiltrating CD19⁺IgM⁺B220^low cells from Tcl1 mice was compared 50 days posttransplantation (shown in [G]) with α-GalCer preloaded (α-GalCer) and unloaded (UnL) Tcl1 CLL cells; healthy wt mice were used as control. Bars indicate means ± SD. **P ≤ .005, ***P ≤ .0005; 1-way ANOVA.