IL-21 in the WM bone marrow microenvironment is derived from activated T cells. (A) PCR for IL-21R, IL-21, and GAPDH was performed on cDNA generated from CD19⁻CD138⁻ cells obtained from the bone marrows of patients with WM (n = 5; nos. 14-18). (B) Additional CD19⁻CD138⁻ samples (n = 3; WM6-WM8) were cultured in the presence or absence of anti-CD3 and anti-CD28 for 72 hours to activate T cells. After total RNA extraction, RT-PCR was performed for IL-21 and GAPDH. (C) Flow cytometric analysis for intracellular IL-21 expression in CD19⁻CD138⁻ WM cells. After culture of CD19⁻CD138⁻ WM cells on plates coated with anti-CD3 in the presence of 1 μg/mL CD28 antibody for 96 hours, cells were stimulated with phorbol myristate acetate, ionomycin, and brefeldin A as described in “Methods.” To assess IL-21 expression in the activated T-cell population, we gated on the CD4⁺ and CD8⁺ populations separately. Shown are histograms representative of 3 different donors. Gray histograms are the respective isotype controls. (D) CD19⁻CD138⁻ cells obtained from patients with WM (n = 3; WM22-WM24) were cultured for 96 hours in the absence or presence of anti-CD3, soluble CD28 antibody (1 μg/mL), or IL-6 (5 ng/mL) for 96 hours. Cell-free supernatants were collected, and the concentration of IL-21 was assessed by ELISA. All experiments were performed in triplicate, and data are represented as mean ± SD. *Statistically significant at P < .05. (E) Costaining of IL-21 with B cells (CD20) and T-cell (CD3) populations in WM bone marrow. Sections were first stained for IL-21 (red), stripped, and restained for either CD3 (blue) or CD20 (green). Digital images from each layer were then overlaid. A representative multicolor composite image is shown.