Background
Umbilical cord blood (UCB) is increasingly used as an alternative source of transplantable CD34
+ haematopoietic stem cells (HSC) for neoplastic and non-neoplastic diseases [
1]. The function of CD34 antigen on human HSC is poorly understood. It has been shown that small interfering RNA-mediated gene silencing of CD34 on human HSC from UCB favours granulocytic and megakaryocytic development at the expense of erythroid commitment, thus shedding light into the potential functional role of this molecule during haematopoietic differentiation [
2]. In recent years, HSC with a CD34
- phenotype have been identified in human UCB, unravelling a hitherto unrecognized complexity within the haematopoietic hierarchy [
3,
4]. Previously, we characterized a rare subpopulation of human UCB CD34
-CD133
-CD7
-lineage
- cells capable of differentiating both into CD34
+CD133
+ HSC in response to stem cell factor (SCF), and into NK/lymphoid progenitors if supported by interleukin (IL)-15 and stromal cells engineered to release human granulocyte colony-stimulating factor (G-CSF) and IL-3 [
5]. In line with this, UCB-derived mesenchymal stem cells have been used to support NK cell expansion induced by the combination of IL-2, IL-3, IL-15 and Flt3-L [
6]. Similarly, Wharton's jelly cells may serve as feeder cells to expand UCB-derived CD34
+ HSC in a potentially clinically applicable culture system [
7]. It should be pointed out that mesenchymal stem cells may activate allogeneic T cells during
in vitro HSC expansion [
8], suggesting need for feeder cell-free culture systems that may support HSC expansion in the absence of untoward effects on other cell types.
IL-21 is a four-helix bundle cytokine released by activated CD4
+ T cells and by NKT cells [
9]. IL-21 signals through a heterodimeric receptor comprising the IL-21 receptor and the common γc of the IL-2 receptor family. IL-21 affects the differentiation and proliferation of NK cells together with IL-2 and IL-15, and is involved in the differentiation of T-helper 17 (Th17) cells, a recently identified subset of CD4
+ T cells that produce IL-17A, IL-17F and IL-22 and promote inflammatory and autoimmune conditions [
10]. In addition, IL-21 suppresses the differentiation of FoxP3-expressing regulatory T cells, leading to enhanced cytotoxic T lymphocyte (CTL) expansion and activity [
11]. Finally, IL-21 is a key regulator of antibody responses against foreign antigens [
12], suggesting that IL-21 may be a master orchestrator of the T-cell-dependent adaptive immune response.
In mice, IL-21 acts in concert with IL-15 to boost the proliferation of both memory and naïve CD8
+ T cells and to foster the
in vitro release of IFN-γ [
13]. Interestingly, IL-21 selectively enhances the effector functions of IL-15-activated murine NK cells, further underpinning the importance of functional interactions between the two cytokines, and mediates potent
in vivo anti-tumour responses [
14]. When provided to serum-replenished cultures of UCB CD34
+lineage
- cells, IL-21 in combination with IL-15, IL-7, Flt3-L and SCF reportedly induces an accelerated NK cell maturation [
15]. Furthermore, IL-21 cooperates with hydrocortisone, IL-15 and Flt3-L in supporting the expansion of NK cells from UCB CD34
+ cells [
16]. However, the contribution of IL-21, if any, to the NK cell differentiation of CD34
-lineage
- cells has not been investigated. It is also unknown whether CD34
-lineage
- cells stimulated with IL-21 may give rise to a qualitatively different NK population when compared to CD34
+ HSC.
The present study aimed to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34-lineage- cells.
Discussion
NK cells are important effectors of the innate immune system and exhibit cytolytic activity against infectious agents and tumour cells. Although our knowledge of NK-cell developmental intermediates remains limited, advances have recently led to a better definition of appropriate culture conditions for the
in vitro generation of mouse and human NK cells from foetal thymus [
27], foetal liver [
28], UCB [
15] and bone marrow HSC [
29]. In early studies, NK-cell development from purified HSC was shown to be stromal-cell dependent [
30]. It has later been demonstrated that the stromal-cell requirements may be replaced by the provision of early-acting cytokines such as SCF, Flt3-L and IL-7 to the cultures [
31]. In particular, SCF and Flt3-L directly induce the expression of IL-2 receptor-β chain on HSC, thereby rendering them susceptible to the NK-cell commitment induced by IL-15 [
31]. Prolonged culture of CD34
+ HSC with IL-15 in the absence of stromal cells can generate pseudo-mature lytic NK cells, e.g., cells expressing markers of mature NK cells (NK1.1 and DX5 in mice, CD56 in humans, CD94-NKG2 receptors in both species) but not Ly49 receptors or KIR [
29,
32]. More recently, UCB CD34
+ cells have been differentiated along the NK lineage with Flt3-L, IL-15, IL-21, and hydrocortisone but in the absence of any stromal cell support [
16]. In addition, IL-21 may synergize with IL-7, IL-15, SCF, Flt3-L and serum supplementation in promoting the generation of NK cells from UCB CD34
+ cells [
15].
The antitumor activity of IL-21 has been demonstrated in murine experimental models where direct effects of IL-21 on NK cells were responsible for tumour suppression [
33]. In addition, the ability of IL-21 to promote long-lasting CD8
+ T-cell-dependent tumour responses has been shown in athymic mice with intraperitoneal or subcutaneous tumours [
34,
35]. IL-21 may also augment human T-cell proliferation driven by polyclonal activation or by a peptide in the absence of other stimuli and may increase CD8
+ T-cell production of IFN-γ induced by IL-15 [
36]. The aforementioned
in vitro and pre-clinical findings have prompted the evaluation of IL-21 as immunotherapy for patients with metastatic melanoma and renal cell carcinoma [
37,
38]. These studies have clearly shown that repeated cycles of IL-21 are well tolerated as an outpatient regimen, thus encouraging further development of IL-21 as an immunotherapy for cancer.
Early studies of UCB transplantation for haematological malignancies have demonstrated an impaired rate and quality of immune reconstitution, which may be associated with an increased rate of infectious complications, particularly at early time points after transplantation [
39]. These clinical observations reinforce the need for novel cell-based therapeutic approaches to overcome the potentially life-threatening infections, including those attributable to a delayed anti-CMV immunity [
40].
We provide evidence that IL-21 favours the NK cell differentiation of CD34
-lineage
- UCB cells in cooperation with IL-15 and in the absence of stromal cell support and serum or hydrocortisone supplementation. The combination of IL-15 and IL-21 displayed a remarkable ability to promote the outgrowth of CD34
-lineage
- cells into NK cells, at variance with IL-21 alone. This is backed by previous observations indicating that the γc-dependent cytokines IL-15 and IL-21 may integrate their signalling and synergise in regulating CD8
+ T-cell expansion and function [
13]. Using murine mature NK cells, Kasaian et al. [
41] have shown that IL-21 may constrain the IL-15-induced expansion of NK cells
in vitro, although their activation status remains unaffected, underpinning the concept that IL-21 may exert diverging effects on murine as opposed to human NK cells. It has also been demonstrated that low doses of IL-21 increase the proliferative response of murine NK cells to either IL-2 or IL-15, whereas high doses of IL-21 may exert an inhibitory effect on NK cell outgrowth [
42]. In our study, IL-21 significantly inhibited the proliferation of CD34
+ cells induced by SCF and Flt3-L, suggesting that IL-21 may also exert opposite effects on HSC proliferation depending on the concomitant cytokine stimulus that is applied.
It should be emphasised that CD34
+ HSC differentiated under the same cytokine conditions expanded more vigorously than their CD34
-lineage
- counterpart. Not unexpectedly, IL-21 promoted the activation of Stat1 and Stat3, but not Stat5 protein, in CD34
-lineage
- cells. NK cells generated
in vitro with IL-15 and IL-21 acquired a CD56
+CD16
-/+ phenotype which differs from the phenotype that we previously observed using Flt3-L, SCF, IL-15 and a stromal feeder layer, where NK progenitor cells stained negatively for CD16 [
5]. The percentage of CD56-expressing CD34
-lineage
- cells was significantly higher compared to that in cultures of CD34
+ HSC, indicating that the former HSC subset has the ability to give origin to a virtually pure NK cell population when confronted with IL-15 and IL-21
in vitro. The natural cytotoxicity receptor NKp46 and the NKG2D antigen were strongly up-regulated on CD34
-lineage
- cells cultured with the combination of IL-15 and IL-21. The activating receptor NKG2D, whose ligands are frequently over-expressed in tumours from multiple origins [
43], could be detected at very low levels in cultures performed with IL-15 alone. Conversely, NKG2D expression levels significantly increased as a result of combined treatment with IL-15 and IL-21, suggesting that the NK cell populations obtained under these culture conditions may represent suitable effectors for cell-based anti-tumour therapeutic approaches. From a molecular standpoint, IL-15 and IL-21 induced mRNA signals for Bcl-2, GATA-3 and for the NK cell-associated transcription factor Id2. Interestingly, NK cell differentiation occurred through a pathway that does not involve TCR rearrangement, indicating that the NK intermediates originating from UCB CD34
-lineage
- cells differ from previously described bi-potent NK/T cells [
27,
28].
Considerable release of IFN-γ only occurred in 4-week old cultures of CD34
-lineage
- cells maintained with IL-15 and IL-21. Conversely, GM-CSF and TNF-α could be detected in supernatants of cultures maintained with either IL-15 alone or IL-15 plus IL-21. Significant secretion of TNF-α could be measured preferentially in cultures stimulated with IL-15 alone. These findings are in good agreement with previous reports on IL-21-induced changes of cytokine secretion by UCB-derived CD34
+ HSC [
16]. In the latter study, IL-21 increased IL-10 and GM-CSF production but lessened TNF-α release after 4-week culturing in the presence of hydrocortisone, Flt3-L and IL-15. CCL3/MIP-1α production occurred under any culture condition herein examined, although the highest production could be documented after challenge with IL-15 and IL-21. The robust GM-CSF and IFN-γ release induced by IL-15 and IL-21 in combination suggests that cytokine-differentiated NK cells may retain the ability to mount effective anti-viral and anti-tumour responses. The significant secretion of CCL3/MIP-1α, a chemokine implicated in the selective mobilization of NK cells from the bone marrow compartment into the peripheral blood [
44], implies that cytokine-matured NK cells may provide
in vivo signals contributing to the regulation of NK homing, retention and migration.
The NK cell populations differentiated with IL-15 and IL-21 were resistant to further maturation with IL-12, as evaluated both in terms of surface membrane phenotype and in terms of cytokine/chemokine release. Specifically, the expression levels of CD16, CD56 and KIR were similar irrespective of the provision of exogenous IL-12. Similarly, CCL3/MIP-1α, TNF-α and GM-CSF release were superimposable in cultures of IL-15+IL-21-differentiated cells that were either stimulated with IL-12 or left untouched.
Finally, the NK cells differentiated with IL-15 and IL-21 underwent exocytosis of secretory granules, as measured by a flow cytometry-based CD107a degranulation assay, upon co-culturing for 4 hours with NK-sensitive tumour cell targets, indicating the acquisition of cytolytic potential. However, the extent of NK granule exocytosis was comparable to that measured in co-cultures of K562 cells and NK cells differentiated from CD34+ HSC with the combination of IL-15 and IL-21.
Authors' contributions
GB carried out the experiments and participated in the design of the study. AM, AP and MC carried out the experiments. GS participated in the design of the study. LP participated in the design of the study. SR participated in the design of the study, carried out the experiments, performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.