Identification of a NCR⁺/NKG2D⁺/LFA‐1^low/CD94⁻ immature human NK cell subset

Abstract

CD56bright natural killer (NK) cells, generated in vitro from CD34 1 hematopoietic progenitor cells, were characterized after a 30-day culture with flt3 ligand plus IL-15. Virtually, all CD56 bright cells expressed CD117, CD25, natural cytotoxicity receptors (NCRs), NKG2D, CD161, and CD244, while only a subset expressed CD18-CD11a (LFA-1), and CD94 molecule, defining an immature CD56 and activatory function that finely control their functional activities. In particular, they express inhibitory receptors for MHC class I (MHC-I) molecules, named killer cell immunoglobulin (Ig)-like receptors (KIRs) and C-type lectin CD94-CD159a, and many triggering molecules like NKp30, NKp44, NKp46, (called natural cytotoxicity receptors, NCRs), NKG2D, CD161, and CD244 (1-4). The majority of peripheral blood (PB) human NK cells are characterized by a phenotype with a low density expression of CD56 (CD56 dim ) and a high expression of CD16 (CD16 bright ), whereas a minority (approximately 5-10%) shows a bright expression of CD56 (CD56 bright ). This latter NK subset presents relatively high expression of some cytokine receptors (CD117 and CD25), lymph node homing receptors (CD62L and CCR7) and the CD94-CD159a heterodimeric inhibitory receptor. Moreover, differently from the well-known CD56 dim /CD16 bright cells, only a small percentage of the CD56 bright subset expresses KIR inhibitory receptors (5-10%) and CD16 dim (20-30%) while, in the majority, these antigens are not expressed (5). On the other hand, CD56bright NK cells are widely expressed in lymphoid tissues (6) and can be generated from CD34 1 cells when cultured with combinations of flt-3 ligand (FL) or stem cell factor plus IL-15 or IL-2 (7-10). During their development, NK cells sequentially acquire many different antigens and some of them allow to distinguish intermediate stages of NK cell differentiation. We had already taken advantage of CD161 antigen expression to define an immature CD161 1 /CD56 2 subset able to kill Jurkat cells via a TRAIL-dependent mechanism (3,11). When cultured with IL-12, this NK cell subset began to express CD56 molecule and became cytotoxic against K562 cells, thus suggesting a

Cytotoxic functions and susceptibility to apoptosis of human CD56bright NK cells differentiated in vitro from CD34+ hematopoietic progenitors

“…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 2 (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.…”

“…The phenotypical and functional characteristics of this subset strongly remind the immature NK Stage 3 classically depicted in lymph nodes by CD117 and CD94 markers (CD117 bright /CD94 2 ) (17) and, more recently in cord and peripheral blood (PB) samples, by the low level of LFA-1 expression (18). The phenotype and the scatter characteristics (small agranular) of this immature LFA-1 low subset of NK cells suggested that, although expressing NKp46 and NKG2D activatory receptors, it could not be still functional due to deficiencies in the cytotoxic machinery (18). With the aim to investigate whether the characteristics of NK cells generated in vitro from CD34 1 progenitors were suitable to counter cancer cells, we have evaluated the sequence through which developing NK cells acquire cytotoxic functions and susceptibility to apoptosis induced via TNF family members.…”

“…Only a pre-treatment with actinomycin D (Fig. 4D), known to inhibit the apoptosis-protection mediated by c-FLIP (3) (12,17,18). In particular, we observed a subset deficient in ORIGINAL ARTICLE b2-integrins, CD94-CD159a, granzyme-B and perforin expression, suggesting that this could represent a stage still unable to perform the cytolytic activity.…”

“…We had already described an immature CD161 1 /CD56 2 subset able to kill only via a TRAIL-dependent mechanism (11,16). The phenotypical and functional characteristics of this subset strongly remind the immature NK Stage 3 classically depicted in lymph nodes by CD117 and CD94 markers (CD117 bright /CD94 2 ) (17) and, more recently in cord and peripheral blood (PB) samples, by the low level of LFA-1 expression (18). The phenotype and the scatter characteristics (small agranular) of this immature LFA-1 low subset of NK cells suggested that, although expressing NKp46 and NKG2D activatory receptors, it could not be still functional due to deficiencies in the cytotoxic machinery (18).…”

Discovering new cell populations

“…Samai et al (4) provide a detailed characterization of maturating human NK cells induced from progenitor CD341 cells using 34 antibodies in three-color staining combinations. The authors show that CD18-CD11a integrin (LFA1) in humans, as well as CD11b in mice, may be useful markers to identify immature stages of NK cell differentiation.…”