Fig 2: 89Zr-CD25 IgG uptake in mouse cells in vitro and in mouse lymphoma in vivo. (A) CD25 expression (left) and anti-mouse 89Zr-CD25 IgG binding (right) in mouse thymus Treg lymphocytes and EL4 mouse lymphoma cells. Cell uptake was compared by using equivalent amounts of cells. Blocking was done by adding 0.5 μM of unlabeled anti-CD25 IgG. (B) Representative coronal (left) and transaxial (right) PET images at 5 days of EL4 tumor-bearing immunocompetent C57/Bl6 mice administered with anti-mouse 89Zr-CD25 IgG alone (top) or co-injected and 0.8 mg of unlabeled anti-CD25 IgG for blocking (middle). Biodistribution data are shown (bottom) as the mean ± S.E of values obtained from two independent experiments (n = 6 per group) N.S., not significant.

Fig 3: Autoradiography and histologic staining of microsections of SUDHL1 tumors from 89Zr-CD25 IgG- injected mice. (Top) Photograph of a film exposed to a microsection of a small tumor showing radioactivity efficiently delivered into the inner tumor regions (left). A microscopic image of the tumor region in the boxed area demonstrates a focus of greater radioactivity (middle) that contained microvessels shown in red by alkaline phosphatase staining in an immediately adjacent microsection (right). (Bottom) Photograph of a film exposed to a microsection of a larger tumor showing greater radiation accumulated in the tumor periphery (left). A microscopic image of the tumor region in the boxed area demonstrates radioactivity distribution (middle) that correlated with immunohistochemistry staining for human CD25 (right).

Fig 4: Circulating free CD25 level and effect on blood 89Zr-CD25 IgG. (A) Western blots of CD25 protein in blood obtained from the heart of 16 SUDHL1 lymphoma-bearing mice (top). Relations of tumor weight with blood 89Zr-CD25 IgG (bottom left) and blood 89Zr-CD25 IgG with blood human CD25 level (bottom right). (B) Relations of tumor weight with 89Zr-CD25 IgG tumor uptake (left) and blood activity (middle) in another group of mice. Western blots and quantified band intensities of tumor CD25 protein according to tumor weight group is shown in the right. Lines were fitted by linear regression and correlation was based on nonparametric Spearman’s analysis. Bars represent the mean ± S.D of four samples per group. N.S., not significant.

Fig 5: CD25 expression and 89Zr-CD25 IgG binding to human lymphoma cells. (A) Western blots of CD25 protein and anti-human 89Zr-CD25 IgG uptakes in H9, Jurkat, and SUDHL1 lymphoma cells (left). Complete blocking of SUDHL1 cell binding by 0.5 μM of unlabeled anti-CD25 IgG (right). (B) Lindmo binding assays for determining the immunoreactive fraction. Five concentrations of SUDHL1 cells were used, from 0.5 to 16 million cells/ml. The final concentration of 89Zr-CD25 IgG was 50 ng/ml, and nonspecific binding was assessed with 5 μg of unlabeled anti-CD25 IgG. A conventional plot of specific and nonspecific binding over total applied radioactivity, as a function of increasing cell concentration is shown (left). A double inverse plot was then drawn using the same data as total applied radioactivity over specific binding, as a function of the inverse cell concentration (right). The immunoreactive fraction was determined through linear extrapolation to the ordinate. Data represent the mean ± S.E of values from two independent experiments [total n = 5 per group; uptake in (A)], or the mean ± S.D of data from a single representative experiment [n = 3 per group; (B)].