To investigate the possibility to develop NK cells suitable to counter cancer cells in oncologic patients, we have evaluated the cytolytic function and susceptibility to apoptosis of human NK cells differentiated in vitro from CD34+ progenitors using cytokine combinations of FLT3-L plus IL-15 with IL-21 (known to generate the CD56dim subset) or without it (known to generate the CD56bright subset) (7–9, 18). First of all, it is important to underline that, upon cytokine stimulation, most of the NK cell markers used to distinguish CD56bright from CD56dim NK cells are either upregulated (CD56, CD16, KIR, CD25, granzymes, perforin, and cytotoxic function) or downregulated (CD62L, CCR7, CD127) (13, 14, 18, 23, 24, and not shown), rendering somehow difficult their reciprocal discrimination. To this regard, in our in vitro experiments, the use of a mAb conjugated with phycoerythrin (typically the brightest fluorochrome for flow cytometry applications) to CD117 (exclusive of CD56bright subset, 5) proved to be the best way to detect CD56bright NK cells and to distinguish them not only from CD56dim/CD117− NK cells but also from CD56+/LFA-1+/Granzyme− monocytes (usually generated in the first 2 weeks of these cultures). Interestingly, while within 20–30 days of IL-21 containing cultures, the highly cytotoxic CD56dim subset died, cultures only with FLT3-L plus IL-15 generated CD117+/CD56bright NK cells that still showed several features of immaturity typical of Stage 3 NK cells (12, 17, 18). In particular, we observed a subset deficient in β2-integrins, CD94-CD159a, granzyme-B and perforin expression, suggesting that this could represent a stage still unable to perform the cytolytic activity. During normal in vivo differentiation, developing NK cells acquire activating and inhibitory receptors and cytotoxic functions in a fashion that prevents NK-mediated auto-aggression against normal cells. One possible explanation to this event is that the expression of functional inhibitory receptors, such as MHC-I receptors or 2B4 molecule on immature NK cells, would precede that of activatory ones (10). Nevertheless, our as well as other reports (7, 10, 17, 18) have already demonstrated both in vitro and in vivo that the expression of activatory molecules, such as NCRs and NKG2D, precede that of MHC-I inhibitory receptors and the inhibitory function of 2B4 on immature NK cells would not explain how CD48− (i.e., non-hematopoietic) autologous cells can be spared by these immature NK cells. NK self-tolerance might be guaranteed by the lack of ligands for NK activating receptors. However, immature myeloid cells, present invivo in the BM (site of NK and erythro-myeloid development) as well as in vitro during NK cell differentiation (10, 11), have shown to express NCRs ligands (25), thus the absence of these ligands could not be guaranteed. For this reason, we propose that, in addition to the inhibitory function of 2B4 molecule (10), immature NK cells would be non-lytic because of the deficiency of β2-integrin adhesion and intragranular cytolytic proteins, thus assuring a fine control of the fail-safe mechanism against all normal self cells. Moreover, MHC-I inhibitory receptors would be expressed as early as NK cells, by upregulating molecules of the cytolytic machinery, become potentially cytotoxic. The reason of an early expression of activatory molecules is not clear, anyway, some evidences support the idea that stimulatory signals are necessary to induce the expression of inhibitory receptors on developing NK cells (reviewed in Ref.15), finally leading to a functionally complete maturation. In our experiments, fully competent and highly cytotoxic NK cells, expressing MHC-I inhibitory receptors, have been obtained after a total of 45 days of IL-15 culture. However, this strong cytolytic function may also depend on a further process of activation induced by the cytokine itself, as suggested by the upregulation of TNF family ligands and receptors in the in vitro developing NK cells. Resembling the intracytoplasmatic TNF-α production (26), the membrane-bound form of TRAIL was observed on both immature (Stage 3) CD56bright/LFA-1− and more mature (Stage 4) CD56bright/LFA-1+ NK cells (18), confirming that TRAIL is a cytotoxic effector molecule expressible also by immature NK cell stages (16). Since immature NK cells are thought to be generated in BM, we had previously hypothesized and demonstrated TRAIL expression in that site (27). Of note, immature erythro-myeloid cells, generated in the BM as well, showed to be sensitive to recombinant TRAIL (27, 28). On the contrary, the highly cytotoxic LAK cells were resistant to apoptosis induced by TRAIL and became significantly susceptible to it only after the down-modulation of the cytokine-dependent c-FLIP (3). Therefore, depending on the cytokine microenvironmental milieu conditions, activated NK cells could develop resistance or sensitivity to negative feedback mechanisms induced by the TNF ligand family members, finally conditioning the length of NK cells response after their activation.
The expression not only of TNF members but also of MHC-I inhibitory receptors, adhesion and cytotoxic effector molecules as well as CD16 and KIR antigens increases with time suggesting that in vitro CD56bright NK cells continue to differentiate/activate from day 30 to day 45 of culture. CD56bright/CD16−/KIR− NK cells have been suggested to differentiate into either CD56dim/CD16+/KIR+ or into CD56bright/CD16+/KIR+ NK cells both in vitro after cytokine stimulation (14, 24, 29, 30) and in vivo in the humanized immune system (HIS) mice model (31) and after HLA-matched hematopoietic stem cell transplantation (32). Similarly, during our secondary culture (from day 30 to 45 of culture), some NK cells begin to express CD16 and KIR. However, the maintenance of CD117 molecule (even though at a lower density than Stage 3, 18) suggests that they would represent fully differentiated/activated CD56bright NK cells rather than CD56dim cells generated from CD56bright ones. Notably, an unusual CD56bright/CD117+/CD16low NK subset, overexpressing intracellular perforin and activating NKG2D and NKp46 receptors, emerges 3 months after hematopoietic stem cell transplantation and its appearance positively correlates with the increased level of IL-15 (32). Moreover, alike to our secondary cultures, Takahashi et al. described the in vitro development of potent effector CD56bright/CD16+ NK cells from CD56bright/CD16− NK cells upon cytokine administration (14).
Although the engagement of CD56 molecule with the FGF-R1 on fibroblasts has been described to induce the direct differentiation of CD56bright to CD56dim cells (33), it is still unclear which growth factors are involved and how they might produce this differentiation without a significant proliferation (29, 33), considering that these two subsets have significantly different telomerase length (30, 33). Moreover, it has been described that human CD56bright NK cells and their mouse counterpart, differently from CD56dim ones, are characterized by the expression of GATA-3 and CD127 (34), suggesting the presence of at least two independent pathways of NK cell differentiation: thymus/lymph node (CD56bright) and bone marrow/spleen (CD56dim). Consistent with this later hypothesis there are evidences of a substantially different activation pathways (CD56bright by cytokines, CD56dim by targets) and thus a different response not only to IL-2 and IL-15 but also to TGF-beta and IL-21 (35–37). In line with this, our data from cultures with IL-21 show a rapid development of highly cytotoxic CD16+/CD56dim/LFA-1+/CD117− NK cells from CD34+ hematopoietic progenitors (8, 9 and Fig. 1) but not from immature (Stage 3) CD56bright NK cells. This cytokine has rather an inhibitory effect on the development of CD56bright NK cells and it actually blocks their differentiation from Stage 3 to Stage 4, thus supporting a model where CD56bright and CD56dim NK cells would represent functionally distinct subsets of mature human NK cells that might diverge earlier than NK cell Stage 3 (12). Alternatively, it could be hypothesized that, starting from CD34+ cultures, IL-21 might induce the generation of FGF-R1 expressing cells able to induce the further differentiation of developing CD56bright cells into CD56dim ones. Standing to this latter hypothesis, however, it is still hard to explain how in IL-21 culture system, the generation of CD56dim NK cells is so short (15–20 days), considering that the generation of CD56bright cells (supposed to mature into CD56dim) takes 20–30 days without IL-21 (7, 9, 18) and, further, that this cytokine inhibits their maturation. Of interest, the CD56dim NK subset, but not the CD56bright one, is significantly decreased in the PB of patients affected by HIV-1, and this positively correlates with the number CD4 T cells. A possible explanation is that the development of CD56dim NK cells would be dependent on IL-21 (9), which is produced by CD4 T lymphocytes (38). In line with this hypothesis, there is the observation that after stem cell transplantation the recovery of CD56dim subset is markedly delayed and resembles the slow reconstitution of CD4+ T cells (39–41). These observations are suggestive of an influence of T cells, at least in the human species, in orienting NK cell progenitors toward CD56dim or CD56bright development.