IL-21 mediates apoptosis through up-regulation of the BH3 family member BIM and enhances both direct and antibody-dependent cellular cytotoxicity in primary chronic lymphocytic leukemia cells in vitro

Abstract

Interleukin-21 (IL-21) is a recently identified gamma-chain receptor cytokine family member that promotes B-cell apoptosis as well as activation of innate immune system. Based on this, we hypothesized that IL-21 might enhance the apoptosis induced by fludarabine and rituximab and also play a role in augmenting immune-mediated clearance of the chronic lymphocytic leukemia (CLL) cells. Our studies demonstrate that the majority of CLL patients have surface IL-21 receptor-alpha, and its expression correlates with apoptosis, tyrosine phosphorylation of STAT1, and up-regulation of the proapoptotic BH3 domain protein BIM. IL-21-induced BIM up-regulation is critical for apoptosis because inhibition of BIM expression using small interfering RNA prevented IL-21-induced apoptosis. IL-21 treatment of CLL cells but not normal T cells with fludarabine or rituximab additively enhanced the direct cytotoxic effect of these therapies. In addition to its proapoptotic effect, IL-21 promoted STAT1 and STAT5 phosphorylation in natural killer cells with concurrent enhanced antibody-dependent cellular cytotoxicity against rituximab-coated CLL cells in vitro. These data provide justification for combination studies of IL-21 with fludarabine and rituximab in CLL and suggest that BIM up-regulation might serve as relevant pharmacodynamic end point to measure biologic effect of this cytokine in vivo.

Figure 1

IL-21–induced cell death in B-CLL cells. (A) Dose kinetics. B-CLL cells isolated from 7 patients were left untreated in media or treated with IL-21 at 0.1, 1, 5, 10, 25, 50, 100, and 200 ng/mL for 72 hours. At 24, 48, and 72 hours, cells were stained with FITC-annexin V and PI and analyzed by flow cytometry. Direct cell death was evaluated by normalizing annexin V/PI–negative cells with the respective media control. The top panel shows a dot plot of one of the representative experiments. The bottom panel shows summarized average of results from the 7 independent experiments. The SD of the mean within the population is shown as error bars (P = .046, implying a slight decrease in the percentage of live cells with dose increases beyond 25 ng/mL). Number on plots are percentages of total cells. (B) Time kinetics. CD19⁺ B-CLL cells were treated with 50, 100, or 200 ng/mL of IL-21 in media, and the direct cell death caused by IL-21 at different time points were assessed by annexin V/PI staining. Direct cell death was evaluated by normalizing annexin V/PI cells with the respective media control. Error bars indicate SD of mean in 16 B-CLL patient cell samples. (C) IL-21–mediated direct cytotoxicity correlates with receptor expression. Primary cell samples from 14 B-CLL patients were stained with PE-labeled mouse mAb to human IL-21R or a nonbinding isotype control and analyzed by flow cytometry. The percentage of the CLL cells expressing IL-21R and percentage of viable CLL cells (annexin/PI compared with media) were analyzed by multiple regression (n = 16; P = .009; R = Pearson correlation). (D) IL-21–mediated direct cytotoxicity in responding patients-dose and time dependence. B-CLL cells were left untreated (media) or treated with IL-21 at 25, 50, 100, and 200 ng/mL for 72 hours. Cells were stained with FITC–annexin V and PI. Direct cell death was evaluated by normalizing annexin V/PI cells with the respective media control. Error bars represent SD of mean in 9 B-CLL patient cell samples (n = 9; P < .01 compared with media control).

Figure 2

Induction of BIM is required for direct apoptosis induced by IL-21. (A) IL-21 induces BIM induction. CD19⁺ B-CLL cells were stimulated with media, IL-21 (100 ng/mL) for 24 hours. Cells were lysed in appropriate buffers and analyzed by Western blotting using specific mAb for Bim and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described in Methods. Figure shows data from a representative responding and nonresponding patient. Numbers below the lanes represent the fold increase in Bim levels in IL-21–stimulated CLL cells relative to unstimulated cells from the same patient and normalized to GAPDH levels. Data are expressed in relative densitometric units. (B) BIM siRNA protects CD19⁺ B-CLL cells from IL-21–mediated direct cytotoxicity. CD19⁺ B-CLL cells were mock transfected (no siRNA) or were transfected with nonsense siRNA or BIM siRNA (n = 3). The CLL cells transfected with Bim siRNA are resistant to the IL-21–mediated direct cytotoxicity compared with scrambled or mock-transfected cells. (C) Western blot analysis of BIM protein expression in nonsense siRNA, BIM-specific siRNA, and mock-transfected CLL cells. The CLL cells mock transfected or nonsense siRNA or BIM siRNA and treated with IL-21 or media therapy for 72 hours and analyzed by immunoblot using BIM specific antibody. GAPDH panel represents the loading control. (D) The bar graph representing the fold increase in the BIM with and without IL-21 is shown. Results shown are representative of 3 independent patients.

Figure 3

IL-21 treatment of CLL cells enhances direct cytotoxicity mediated by antibodies and fludarabine. (A) IL-21 mediates tyrosine phosphorylation of STAT1^Y701 and STAT3^Y705 in C19⁺ B-CLL cells. CD19⁺ B-CLL cells were stimulated with media or IL-21 (100 ng/mL) for 24 hours. Cells were lysed in appropriate buffers and analyzed by Western blotting using mAb specific for indicated STAT proteins and GAPDH. Shown are the results of a representative responding and nonresponding patient. (B) IL-21 enhances additively antibody and fludarabine-mediated cytotoxicity. CD19⁺ B-CLL cells were treated with media or IL-21 at 100 ng/mL concentration for 18 hours followed by incubation with cross-linker alone (media), trastuzumab with cross-linker (Trastuzumab), rituximab with cross-linker (Rituximab), alemtuzumab with cross-linker (Alemtuzumab), and fludarabine. At 48 hours, direct cytotoxicity by antibody exposure and fludarabine was analyzed by annexin V/PI staining. Shown here are the results from 7 consecutive experiments. Error bars are SD among samples. (C) IL-21 enhances fludarabine-induced cytotoxicity. Dose kinetics. CD19⁺ B-CLL cells were treated with IL-21 at 100 ng/mL concentration of IL-21 or media alone for 18 hours, and the pretreated cells were incubated with fludarabine at 0.1, 1, 2.5, and 5 μM concentration. At 48 hours, direct cytotoxicity by fludarabine was analyzed by annexin V/PI staining. Shown here are the results from 7 consecutive experiments. Error bars are SD between samples.

Figure 4

IL-21 treatment of T cells does not enhance direct cytotoxicity of fludarabine. CD3⁺ T cells from CLL patients were treated with IL-21 at 100 ng/mL concentration or media alone and at indicated concentration of fludarabine ranging from 0.08 to 20μM. At 72 hours, direct cytotoxicity by fludarabine was analyzed by annexin V/PI staining. Shown here is the result from 5 consecutive experiments. Error bars are SD between samples.

Figure 5

IL-21 enhances NK-mediated ADCC of rituximab-coated CLL cells. (A) IL-21 mediates tyrosine phosphorylation of STAT1 and STAT5 in NK cells. NK cells (CLL patient derived) were stimulated with media, IL-21 (10 ng/mL), IL-2 (10 U), or IFN-α (10⁴ U/mL). Cells were permeabilized and stained for p-STAT1 and p-STAT5 with appropriate controls as described in “Assessment of STAT and STAT3 signaling by flow cytometry” and analyzed by flow cytometry. Shown is a representative histogram of 3 independent experiments. (B) IL-21 enhances autologous NK cell–mediated ADCC of rituximab-coated CLL cells. ⁵¹Cr-labeled freshly isolated B-CLL cells were incubated with alemtuzumab, rituximab, or isotype control trastuzumab. CLL patient–derived NK cells treated with media alone (M) or 100 ng/mL of IL-21 (E) were incubated with autologous B-CLL cells at 37°C for 18 hours. Percentages of relative cytotoxicity were measured after 4 hours as described in “Antibody-dependent cellular cytotoxicity assay.” Data shown here are summary of 3 patient samples, and error bars represent SD between patients. IL-21 plus rituximab significantly enhances ADCC compared with rituximab alone (P < .001 at all E:T ratios). (C) IL-21 enhances allogeneic NK cell–mediated ADCC of rituximab-coated CLL cells. Freshly isolated CD19⁺ B-CLL cells were incubated with alemtuzumab, rituximab, or isotype control trastuzumab. Donor CLL patient–derived NK cells were treated with media alone or 100 ng/mL of IL-21 (18 hours) and incubated with B-CLL cells at 37°C. Percentages of relative cytotoxicity was measured after 4 hours as described above. Data shown here are a summary of 3 patient samples, and error bars represent SD between patients (n = 3). IL-21 plus rituximab significantly enhances ADCC compared with rituximab alone (P = .04 at an E:T ratio of 25:1).