Differential effects of IL-2 and IL-21 on expansion of the CD4+CD25+Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia

CD4+ CD25+ regulatory T cells are expanded in solid and hematological malignancies including Chronic Lymphocytic Leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the novel common gamma chain cytokine interleukin- 21 (IL-21) on primary B cells from CLL patients. Since members of this family of cytokines are known to exhibit their effects on diverse immune cells, we have examined the effects of IL-21 on CLL patient derived regulatory T cell induction, expansion and the inhibitory effect on natural killer cells in vitro. We demonstrate here the expression of IL-21 receptor in CD4+CD25High regulatory cells from CLL patients. In contrast to IL-2, the IL-21 cytokine failed to mediate expansion of regulatory T cells or induced expression of Foxp3 in CD4+CD25Intermediate or CD4+CD25Dim/- T cells in whole blood derived from CLL patients. Interestingly, in contrast to their differential effects on expansion of the CD4+CD25+Foxp3+T cells, IL-2 and IL-21 exhibited a redundant role in T-reg mediated suppression of NK cell mediated antibody dependent cytotoxicity function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy.


Introduction
T regulatory cells (Tregs) are CD4 + CD25 High T cells identified by their expression of forkhead transcription factor (Foxp3). 1 Foxp3 is responsible for the acquisition of immunosuppressive function of Tregs and for their anergy. Tregs exhibit immuneregulatory properties and their dysfunction has been implicated in the development of allergic and autoimmune diseases. 2 The increased numbers of Tregs are observed in several malignancies and have been implicated in additional mechanisms reducing immunity, which may contribute to poor prognosis and responses to chemotherapy. 3,4 The number and inhibitory functions of the Tregs can be decreased by chemotherapeutic agents such as fludarabine therapy in chronic lymphocytic leukemia (CLL). 5 Tregs can develop both in the thymus and periphery. Tregs that develop in the thymus are called natural Tregs (nTregs) and those that CD4 + CD25 + regulatory t cells are expanded in solid and hematological malignancies including chronic lymphocytic leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the novel common gamma chain cytokine interleukin (IL)-21 on primary B cells from CLL patients. Since members of this family of cytokines are known to exhibit their effects on diverse immune cells, we have examined the effects of IL-21 on CLL patient derived regulatory t cell (treg) induction, expansion and the inhibitory effect on natural killer cells in vitro. We demonstrate here the expression of IL-21 receptor in CD4 + CD25 High regulatory cells from CLL patients. In contrast to IL-2, the IL-21 cytokine failed to mediate expansion of regulatory t cells or induced expression of Foxp3 in CD4 + CD25 Intermediate or CD4 + CD25 Dim/t cells in whole blood derived from CLL patients. Interestingly, in contrast to their differential effects on expansion of the CD4 + CD25 + Foxp3 + t cells, IL-2 and IL-21 exhibited a redundant role in treg mediated suppression of NK cell mediated antibody dependent cytotoxicity function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy. Differential effects of IL-2 and IL-21 on expansion of the CD4 + CD25 + Foxp3 + T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia Aruna Gowda,1 Asha Ramanunni, 1 Carolyn Cheney, 1 Darlene Rozewski, 1 Wayne Kindsvogel, 2 Amy Lehman, 3 David Jarjoura, 3 Michael Caligiuri, 1 John C. Byrd 1, * and Natarajan Muthusamy 1, * 1 are actively generated in the periphery through the conversion of CD4 + CD25 -T cells into Foxp3 + cells are known as induced Tregs. 6 Although increased Tregs is a characteristic feature in many tumors, the molecular and cellular basis for their increase and maintenance is not clearly understood. 3 Interleukin (IL)-2 is a member of the common gamma chain family of cytokines with potent T cell growth promoting activity that has been used clinically to enhance T cell immunity in patients with acquired immune deficiency syndrome or cancer. Interestingly, disruption of the IL-2 pathway results in lymphoid hyperplasia and autoimmunity rather than immune deficiency, indicating that the major physiological function of IL-2 is to limit rather than enhance T cell responses. This is mediated by the critical role of IL-2 in the development and peripheral expansion of CD4(+)CD25(+) regulatory T cells. The role of common gamma chain containing cytokines such as IL-2 and IL-21 on that IL-21 is not an essential cytokine to Treg cell expansion, but may have a redundant role in Treg cell function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy.

CD4 + CD25 + regulatory T cells from CLL patients express the IL-21 receptor.
We and others have recently demonstrated the direct cytotoxic effect of IL-21 on CD19 + primary B cells from CLL patients. 10,11,13 While common gamma chain cytokines such as IL-2 and IL-21 have been shown to exert diverse effects on multiple cell types including B cells, T cells and NK cells, little is known of their effect on CD4 + CD25 High Tregs from CLL patients. As the IL-21 receptor expression has been observed in B cells and CD4 + T cells, we first examined whether the CD4 + CD25 + Treg cells isolated from CLL patients express IL-21 receptor. Of the six CLL patient samples examined, the IL-21 receptor was observed in five patients. Interestingly, differential levels of IL-21 receptor expression were observed in CD4 + CD25 High , CD4 + CD25 Intermediate and CD4 + CD25 Dim/populations. Thus, majority of CD4 + CD25 High regulatory T cells (68% to 95%) expressed high levels of IL21 receptor (MFI > 400; ranging from 400-900), while fewer CD4 + CD25 Intermediate (13-29%) and CD4 + CD25 Dim/-(1-5%) T cells expressed high levels of IL-21 receptor (Fig. 1).

IL-2 but not IL-21 causes expansion of the CD4 + CD25 + T cells. Several studies have revealed an essential role for IL-2
Treg biology is diverse. Several studies have suggested that IL-2 has a non-redundant function in the generation of Treg in the thymus. In its absence, Tregs failed to survive and expand in the periphery, and so IL-2 signaling in these cells appears to be critical for their homeostasis. 7 IL-21, a novel common gamma chain family member, is also a Type I cytokine secreted by activated T cells. IL-21 causes a broad range of effects, including enhancement of the proliferation of T cells, differentiation of B cells into plasma cells and augmentation of the cytolytic function of natural killer cells. 8,9 It also plays a role in the development of autoimmunity and exhibits anti-tumor activity. We and others have shown direct cytotoxic effect of IL-21 on a subpopulation of primary B cells from CLL patients through activation of STAT1 signaling events and upregulation of the BH3-only containing, proapoptotic protein BIM. 10 Although IL-2 and IL-21 share a common gamma chain receptor and its downstream signaling pathways, they exhibit differential effects on diverse immune cells. Thus, in addition to its activities in normal lymphoid cells, IL-21 is an in vitro growth factor for myeloma and acute-T cell leukemia cells; however, it induces apoptosis of CLL B cells. [10][11][12] In the present study, we demonstrate that CD4 + CD25 + Treg cells from CLL patients express the IL-21 receptor. Interestingly, in contrast to IL-2, we report that IL-21 does not expand the Treg cell population or increase the expression of Foxp3 in CD4 + CD25 Intermediate T cells derived from whole blood of CLL patients. In contrast to their differential effects on Treg expansion, IL-2 and IL-21 mediate redundant roles in CD4 + CD25 bright Treg mediated suppression of natural killer cell (NK) mediated antibody-dependent cellular cytotoxicity (ADCC). These observations suggest  the suppression was augmented by CD3 activation, IL-2 or IL-21 treatment (Fig. 3). Overall, these results have revealed a differential role for IL-2 and IL-21 in CD4 + CD25 bright FoxP3 + cells expansion, and a redundant role in suppression of NK mediated ADCC functions.

Discussion
We and others have recently demonstrated potential use for IL-21 as a cytotoxic agent in CLL. 10,12 Given the pleiotropic effects of the common gamma chain family of cytokines such as IL-2 and IL-21 on B, T and NK cells, we examined the effects IL-2 and IL-21 on regulatory T cell homeostasis and NK mediated ADCC function. We observed that regulatory T cells from majority of CLL patients variably express the IL-21 receptor. In contrast to IL2, IL-21 failed to induce the expansion of regulatory T cells as evidenced by lack of increase in CD4 + CD25 + Foxp3 + T cells with IL-21. In contrast to their differential effect on expansion of the CD4 + CD25 + Foxp3 + T cells, IL-2 and IL-21 exhibited a redundant role in suppression of NK cell mediated ADCC function.
The differential effects of IL-2 and IL-21 is an interesting phenomenon that has been observed previously in NOD mice, wherein CD8 + autoreactive T cells are expanded with high serum IL-21 levels while the CD4 + CD25 + regulatory T cells are decreased in these mice, suggesting that the IL-2, but not IL-21, is an essential cytokine for Treg cells. 18,19 At higher concentration of IL-21 we have observed expansion of Tregs in some patients, which is probably related to the co-culturing of the regulatory cells with other T cells and B cells in whole blood wherein IL-21 could have activated the CD8 + CD25 -T cells to secrete IL-2, resulting in expansion of Treg cells in some patient samples. The novel method of studying the effects of IL-2 and IL-21 in an in vitro setting using whole blood, thus simulating physiological conditions where there is complex interaction of immune cells and CLL cells, is highly relevant and clinically applicable. In this context, we speculate that IL-21 is likely to mediate limited effects on Treg cell growth and expansion in CLL patients in vivo. Further, in line with the other studies, IL-21, in contrast to IL-2, did not expand the CD4 + CD25 Intermediate population or enhance Foxp3 expression in the CD4 + CD25 Dim/-T cells, suggesting that the IL-21 is not critical for the homeostasis of Tregs, and thus may not result in the increased inducible Treg cells in periphery observed with IL-2.
Treg cells suppress the proliferation of cytotoxic T cells and inhibit the secretion of interferon gamma by NK cells. 8 However the role played by Tregs in inhibition of NK cell mediated ADCC has not been previously described. The studies described here demonstrate that Tregs can actively inhibit the ADCC function of NK cells when both are co-cultured for 24 hours. Interestingly, the naïve Treg cells exhibited suppressive effects even when not pre-activated with cytokines or anti-CD3 antibody (Fig. 3A). The presence of plate bound anti CD3 significantly enhanced this suppressive effect (Fig. 3B). This is consistent with studies where Treg cells are required to be activated through the CD3 and CD28 to exhibit suppressive function. 20 Freshly separated naïve Tregs have been shown to suppress direct NK cytotoxicity in the survival, expansion and homeostasis of Treg cells. 4 IL-21 is a member of the common gamma chain receptor family of cytokines that shares structural and signaling features through the JAK/STAT pathway. One of the impediments identified in clinical use of IL-2 was ascribed to expansion of Treg populations. 14 In order to determine if IL-21 modulates Treg homeostasis in CLL patients, CLL patient derived whole blood was treated with varying concentrations of IL-2 or IL-21 for 48 hours. The PBMCs were then isolated and examined by three color flow cytometry for CD4 + CD25 + T cells expressing intracellular Foxp3. Pretreatment of whole blood with concentrations of IL-2 or IL-21 with as low as (10 ng/ml) resulted in expansion of CD4 + CD25 High Foxp3 + cells selectively with IL-2, but not with IL-21 ( Fig. 2A and B). Even increasing the concentration of IL-21 to ten fold failed to result in increased CD4 + CD25 High Foxp3 + cells (data not shown).
To investigate if IL-2 and IL-21 differentially modulated Fox3p expression in CD4 + CD25 Intermediate and CD4 + CD25 Dim/-T cell populations, we analyzed the expression of Foxp3 in these populations by multi-color flow cytometry. IL-2 had moderate effect on the increase in Foxp3 expressing cells in the CD4 + CD25 Intermediate cohort ( Fig. 2C and D). In the CD4 + CD25 Dim/-T cells ( Fig.  2E and F) IL-2 increased the Foxp3 expressing cells by two fold at both the low and high concentrations. In contrast, IL-21 failed to increase the number of CD4 + CD25 Intermediate Foxp3 + or CD4 + CD25 Dim/-Foxp3 + T cells. When the IL-2 and IL-21 effects were compared independently to the untreated controls, the expansion of CD4 + CD25 High cells was significant only with IL-2 (estimated difference 1.39, CI 0.63, 2.14, p = 0.0057), but not with IL-21 treatment (estimated difference 0.75, CI -0.01, 1.15 and p = 0.1048).
IL-2 and IL-21 mediate redundant roles in CD4 + CD25 bright treg mediated suppression of natural killer cell mediated antibody dependent cellular cytotoxicity. Several studies have demonstrated the ability of Tregs to suppress direct killing by NK cells. However, no evidence has been linked to suppressive activity of NK mediated ADCC by Tregs. 15 Given the importance of ADCC mediated by therapeutic antibodies such as rituximab in CLL, we next assessed the impact of IL-21 or IL-2 on the inhibitory effect of Tregs on ADCC function. Previous reports have shown minimal suppressive function by freshly separated naïve Treg in vitro, and a need for anti-CD3 mediated activation to enhance their suppressive effect. 16,17 In order to assess the role of IL-2 or IL-21 on the Treg function in NK cell mediated ADCC, we plated the CD4 + CD25 high FoxP3 + cells in the presence of media, IL-2 or IL-21 with or without plate-immobilized anti-CD3 antibodies for 72 hours. As shown in Figure 3A, naïve Tregs mediated ∼50% reduction in CD20 directed rituximab mediated ADCC against Raji target cells. Consistent with previous reports, compared to naïve cells, the Treg cells activated with immobilized anti-CD3 (Fig. 3B), when co-cultured with the NK cells for 24 hours strongly suppressed rituximab mediated ADCC ( Fig. 3A and B). Most importantly, IL2 or IL-21 treated Treg cells exhibited suppression of NK mediated ADCC, comparable to that seen with anti-CD3 activated Tregs (Fig. 3B-D). Thus, the naïve Tregs suppressed NK cell mediated ADCC, and when cultured together for 4 hours. 21,22 In our experiments, the Treg mediated suppression of the ADCC function of the NK cell line was evident at 1:1 ratio without CD3 activation, but required 72 hours of culture. In previously published results, these cells have been shown not to secrete any cytokines and their suppression to be mediated through TGFbeta. 19 Interestingly, the suppression of ADCC by Tregs was enhanced by both IL-2 and IL-21 to a similar extent, and by CD3 activation. This is most likely ascribed to the fact that both these cytokines that belong to the common gamma chain and may share redundant signaling features. Despite these similarities, and in contrast to IL-2, IL-21 lacks the ability to expand CD4 + CD25 High cells, and importantly, prevents the intracellular Foxp3 acquisition by CD4 + CD25 -T cells that is essential for suppressive function. Furthermore, the high concentrations of IL-21 with reduced Treg cell population in NOD mice further support our findings that IL-21 may not be a critical cytokine for the homeostasis of Treg cells in these mice.
The studies herein, which show that IL-21 does not induce expansion of Treg cells in CLL patients, have tremendous clinical relevance. Thus, recent observations on an IL-21 mediated direct apoptotic effect that functions through induction of pro-apoptotic Bim protein and activation of granzyme B in CLL cells while activating and recruiting the innate immune system for tumor clearance has tremendous clinical implications. 10,12 Clinically, IL-21 therapy as single agent, or in combination with rituximab, or rituximab and fludarabine in patients with CLL, is highly feasible. Given the tolerable toxicities with IL-21, in contrast to IL-2 which has significant infusional toxicities and pro-survival effect on CLL cells, our results provide a compelling (50 µg/10 8 cells) and subjected to magnetic columns. Purity of the enriched cells ranged from 85-90%. The cells were activated in RPMI 1640 supplemented, with penicillin (56 U/ml), streptomycin (56 µg/ml) (all from Invitrogen, Carlsbad, CA) and 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT) in the presence or absence of indicated concentrations of IL-2 or IL-21 at 37°C, in an atmosphere of 5% CO 2 for indicated time points.
Antibody dependent cellular cytotoxicity (ADCC) inhibition assay. The Treg cells from normal healthy volunteer donor were co-incubated in fresh media without the cytokines with NK-92 cells, at a 1:1 concentration for 24 hours. The NK-92 cell line is an IL-2 dependent human NK cell clone derived from human non-Hodgkin's lymphoma with large granular lymphocyte phenotype (CD56 + CD16 -) which has been transfected with CD16. 25 For the ADCC experiments these cells were starved of IL-2 for 48 hours prior to use. Raji cells were used as the targets in the standard ADCC assay at indicated effector: target (E:T) ratios. ADCC activity was determined by standard 4-hour 51 Chromium-release assay as described previously. 26 Effector cells (NK-92 cells) were co-cultured with CD4 + CD25 + Treg cells for 24 hours at 1:1 ratio. 51 Cr-labeled target cells (Raji cell line) were incubated with indicated antibodies for 20 minutes, washed off excess antibody and the cells were placed in 96-well plates at the indicated effector to target ratio (E:T). After 4-hour incubation, supernatants were harvested and counted on a gamma counter. The percentage of relative lysis was determined by: % lysis = 100 x (ER-SR)/(MR-SR) where ER, SR and MR represent experimental, spontaneous and maximum 51 Chromium release respectively.