HUMAN NK cells are characterized by NK cell receptors (NKRs) with inhibitory 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 (CD56dim) and a high expression of CD16 (CD16bright), whereas a minority (approximately 5–10%) shows a bright expression of CD56 (CD56bright). 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 CD56dim/CD16bright cells, only a small percentage of the CD56bright subset expresses KIR inhibitory receptors (5–10%) and CD16dim (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+ 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+/CD56− 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 strict correlation between CD56 surface expression and the ability to perform Ca++-dependent lysis of target cells (3). Nevertheless, the presence of a heterogeneous subpopulation within the CD56+ NK cells generated in vitro is suggested by the coexistence of CD56+ subsets able to produce different cytokines (12). More recently, CD117 and CD94 markers have been used to distinguish the immature CD117bright/CD94− NK subset (stage 3 NK cells) from its mature (stage 4) CD117low/CD94+ progeny (13, 14). It should be noted that IL-15 and IL-2 induce both differentiation and activation of NK cells, therefore in vitro differentiated NK are also activated. Upon cytokine stimulation, NK cells become lymphokine-activated killer (LAK) cells that proliferate, produce several cytokines and upregulate many intracellular and surface molecules (1, 5, 15–18). After binding to target cells, mainly mediated by β2-integrins (CD11/CD18) and CD2 adhesion molecules (18), NK/LAK cells can perform spontaneous cytotoxicity against target cells without prior sensitization (1, 11, 15, 16, 19). There is still limited knowledge on differentiation antigens able to identify immature human NK cells and the specific sequence through which developing NK cells acquire the expression of NKR and other NK molecules (4, 20, 21). To this aim, we have evaluated the expression of specific surface antigens during in vitro development of NK cells generated from CD34+ PB hematopoietic progenitors in culture with IL-15 plus FL. In the present report, we demonstrate that LFA-1 is a useful marker to distinguish an immature stage of the CD56bright NK subset.
CD56bright natural killer (NK) cells, generated in vitro from CD34+ hematopoietic progenitor cells, were characterized after a 30-day culture with flt3 ligand plus IL-15. Virtually, all CD56bright 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 CD56bright/NCRs+/NKG2D+/LFA-1−/CD94− subset. Another small subset of cells expressing CD94 but not LFA-1 integrin was also identified, suggesting that during NK differentiation LFA-1 might be upregulated later than CD94. To verify this hypothesis in vivo, we evaluated the NK cell expression of LFA-1 in both peripheral and umbilical cord blood samples. Interestingly, in these blood fluids, we have identified a lineage negative CD34−/LFA-1low/NKp46dim/NKG2Ddim/CD94− subset that resembled an immature stage of NK cells present in lymph nodes. Altogether, the results indicate that CD18-CD11a integrin, as well as CD11b in mice, may be a useful marker to identify immature stages of NK cell differentiation. © 2009 International Society for Advancement of Cytometry
MATERIALS AND METHODS
Reagents and Antibodies
Monoclonal antibodies (mAbs) for flow cytometry were from different companies (see Table 1 for specifications). Briefly, mAbs to CD56, CD16, CD2, CD8, CD11a, CD11b, CD11c, CD18, CD25, CD3, CD19, CD34, and CD117 were from Caltag Laboratories/Invitrogen (Burlingame, CA); to CD123, CD94, CD161, CD158a,b (recognized by the HP-3E4 and CH-L clones), NKG2D, CD244, CCR7, CD7, and CD122 were from BD Biosciences Pharmingen (San Jose, CA); to CD5, CD33, CD159a, NKp30, NKp44, and NKp46 were IOTest-type, from Beckman Coulter (Fullerton, CA); to HLA-DR, and CD62L were from Ancell (Bayport, MN); to TRAIL was from Alexis Biochemicals (Gruenberg, Germany); to CD158e (NKB1) was from BioLegend (San Diego, CA). Anti-CD158e biotinylated antibody was used in combination with streptavidin PE (Caltag Laboratories/Invitrogen). TRAIL staining was performed with a three-step procedure using purified anti-TRAIL, then goat anti-mouse IgG-PE (Caltag Laboratories/Invitrogen) and, finally, directly conjugated mAbs. MAb to CD244 (IgM, clone CO54) and goat anti-mouse IgM-PE were kindly provided by Prof. A. Moretta (4). Recombinant IL-15 was purchased from Peprotech EC (London, UK), and FL from R&D Systems (Minneapolis, MN).
|CD2||Caltag Laboratories/Invitrogen||S5.5||IgG2a||FITC, PE|
|CD5||IOTest, Beckman Coulter, Inc.||BL1a||IgG2a||PE|
|CD7||BD Bioscience Pharmingen||M-T701||IgGl||PE|
|CD8||Caltag Laboratories/Invitrogen||3B5||IgG2a||FITC, PE|
|CD16||Caltag Laboratories/Invitrogen||3G8||IgGl||PE, TC|
|CD33||IOTest, Beckman Coulter, Inc.||D3HL60.251||IgGl||PE|
|CD34||Caltag Laboratories/Invitrogen||S81 class III||IgGl||PE,TC|
|CD56||Caltag Laboratories/Invitrogen||MEM-188||IgG2a||FITC, PE, TC|
|CD94||BD Bioscience Pharmingen||HP-3D9||IgGl||PE|
|CD122||BD Bioscience Pharmingen||Mik-β2||IgG2a||PE|
|CD123||BD Bioscience Pharmingen||9F5||IgGl||PE-Cy5|
|CD158a||BD Bioscience Pharmingen||HP3E4||IgM||FITC, PE|
|CD158b||BD Bioscience Pharmingen||CH-L||IgG2b||FITC, PE|
|CD159a (NKG2A)||IOTest, Beckman Coulter, Inc.||Z199||IgG2b||PE|
|CD161||BD Bioscience Pharmingen||DX12||IgGl||PE|
|CD244 (2B4)||a gift of Prof. A. Moretta (4)||C054||IgM||Supernatant|
|CD244 (2B4)||BD Bioscience Pharmingen||2-69||IgG2a||FITC|
|CCR7 (CD197)||BD Bioscience Pharmingen||3D12||IgG2a||PE|
|NKG2D||BD Bioscience Pharmingen||1D11||IgGl||PE|
|NKp30 (CD337)||IOTest, Beckman Coulter, Inc.||Z25||IgGl||PE|
|NKp44 (CD336)||IOTest, Beckman Coulter, Inc.||Z231||IgGl||PE|
|NKp46 (CD335)||IOTest, Beckman Coulter, Inc.||BAB281||IgGl||PE|
|TRAIL (CD253)||Alexis Biochemicals||2E5||IgGl||Purified|
|Mouse isotype controls||Caltag Laboratories/Invitrogen||IgGl||Purified, Biotin, FITC, PE, TC|
|Mouse isotype controls||Caltag Laboratories/Invitrogen||IgG2a||FITC, PE, TC|
|Mouse isotype controls||Caltag Laboratories/Invitrogen||IgG2b||FITC, PE|
|Mouse isotype controls||Caltag Laboratories/Invitrogen||IgM||Purified, FITC, PE|
Cell Isolation and Culture
Mononuclear cells from PB buffy coats were provided by the Transfusion Centre of Urbino Hospital. Immunomagnetic positive selection of CD34+ cells was performed from unmobilized PB samples using the CD34 isolation kit and the Vario-MACS magnetic cell sorting program (Miltenyi Biotec, Auburn, CA) in accordance with the manufacturer's recommendations. Purity was checked by flow cytometry on an aliquot of the population selected using anti-CD34-PE, (HPCA-2, BD Biosciences Pharmingen, which recognizes a class III epitope of the CD34 molecule). Only samples with a purity >95% were used for in vitro NK cell differentiation. Purified CD34+ cells were cultured for 15–30 days in 1–2 ml X- Vivo 20 medium (BioWhitakker, Walkersville, MA) supplemented with 10% human AB serum, FL (20 ng/ml) and IL-15 (20 ng/ml). Cytokines and medium were replaced every 4 days during the culture period. To obtain higher percentages of CD56bright cells (>90%), FL administration was stopped after 15 days of culture. For comparison with NK cells generated from CD34+ cells, in some experiments, after the positive selection of CD34+ cells, CD56+ NK cells (purity > 95%) were purified by negative selection from the eluted lymphocytes using the Vario-MACS and the NK isolation kit II (Miltenyi Biotec) and then cultured with IL-15 (20 ng/ml). Considering that the receptor for FL (CD135) is not expressed by mature NK cells, most of the experiments regarding mature NK cells were performed without the use of this cytokine. When indicated, lineage negative (Lin−)/CD34− NK cells from PB or umbilical cord blood (UCB) samples were analyzed for surface antigens. Briefly, mononuclear cells were stained with a cocktail of anti-CD3, -CD19, -CD16, -CD123, and -CD34 PE-Cy5-conjugated mAbs used as a lineage exclusion channel (dump channel). PE-Cy5-positive cells (i.e., T, B, CD56dim NK cells, basophils, and hematopoietic progenitors) were electronically gated out to select the remaining CD56bright/Lin−/CD34− NK subset. To focus on an immature LFA-1low subset, we performed a gate based on scatter characteristics of LFA-1low cells (backgating using LFA-1low positive cells).
Flow Cytometric Surface Antigen Detection
Three-color immunofluorescence analyses were performed with the indicated mAbs gating on the lymphoblastic or lymphocytic region. Fluorescein isothiocyanate (FITC)-, PE-, or PE-Cy5 labeled mAbs were used to detect the surface antigens. Quadrants were set based on background staining (<1-2%) with isotype control mAbs, except in Figure 3 where the quadrants in the x-axis (CD18) were set in order to distinguish CD18 low and high expression. In Figures 1 and 2, to focus on NK cells and reduce the background of isotype match control mAbs (mainly produced by contaminating myeloid cells), we performed a gate based on the scatter characteristics of CD56+ cells (backgating using CD56+ cells). Single-stained controls with anti-CD45 were used to set instrument compensation. Samples were acquired on a FACScan or a FACScalibur flow cytometer (Becton-Dickinson BD Biosciences, San Jose, CA). Flow cytometry data were analyzed using WinMDI software.
Percentages of cells expressing selected NK antigens were calculated at different time points during NK cell differentiation as mean values ± their standard deviations (mean ± SD). To explore the existence of statistically significant differences, the two-tailed, two-sample Student T test was used. P values <0.05 were considered statistically significant.
Characterization of Surface Phenotype of CD56bright NK Cell Subset Derived from a 20–30 Days Culture of Purified CD34+ Hematopoietic Progenitors
Hematopoietic progenitors cultured in the presence of FL plus IL-15 differentiate into cytotoxic and cytokine-producing CD56bright NK cells (7, 8, 10–12). To identify intermediate stages of NK differentiation, the expression of several surface markers on CD56bright cells was evaluated.
After 20–30 days of culture, the majority of CD56bright NK cells, like their resting PB counterpart, coexpressed NK activatory molecules (NCRs, NKG2D, CD161, CD244) and cytokine receptors (CD122, CD25, CD117) (Fig. 1 and Table 2). On the contrary, lymph node homing receptors (CD62L, CCR7), HLA-DR activation marker, adhesion molecules (CD11a, CD11b, CD11c, CD2), NK inhibitory receptors (KIRs, CD94), and other NK markers (CD16, CD8) were present only on a cell subset or were undetectable (Fig. 1 and Table 2).
|Percent within CD56+ cells|
|Antigena||Day 20 mean SD(n)b||Day 30 mean SD(n)|
|CD11a||34 ± 13 (5)||66 ± 19 (7)|
|CD94||60 ± 15 (5)||85 ± 13 (7)|
|CD25dimc||>90% (1)||>90% (4)|
|CD117dim||>80% (1)||>80% (5)|
|CD122 dim||>90% (2)||>90% (3)|
|CD161dim||>90% (3)||>90% (3)|
|NKG2Ddim||>90% (1)||>90% (4)|
|NKp30 (CD337)||>95% (2)||>95% (8)|
|NKp44 (CD336)||>95% (2)||>95% (7)|
|NKp46 (CD335)||>95% (5)||>95% (7)|
|CD244(2B4)||>95% (2)||>95% (3)|
Considering that IL-15 induces both differentiation and activation, we evaluated the CD56bright phenotype after a short-term (3–5 days) IL-15 culture of purified PB NK cells. IL-15-stimulated cells expressed low levels of CD62L and CCR7 and a variable expression of CD11b, suggesting an in vitro down-modulation of these IL-15-regulated molecules (6, 22 and data not shown). Differently from the aforementioned markers, CD11a-CD18 heterodimer (LFA-1), CD11c, CD2, and CD94 maintained high expression levels on the majority of IL-15 stimulated cells, suggesting a relatively immature phenotype within the in vitro differentiated NK population.
Characterization of LFA-1−/CD56bright NK Subset Obtained in Culture
Although NK activatory molecules and cytokine receptors were expressed as early as CD56 antigen, the percentages of CD11a and CD94 antigens within CD56+ cell population progressively increased during the 30-day culture (see Table 2). Interestingly, CD56bright/CD11a+ cells were at percentage significantly lower than CD56bright/CD94+ cells (Fig. 1 and Table 2), suggesting that LFA-1 could be upregulated relatively later than CD94. As a matter of fact, a small percentage of CD56bright cells expressed CD94 (at low density) but not CD18 antigen (Fig. 2). As expected, expression profiles of CD11a and CD18 completely overlapped, since together they constitute LFA-1 heterodimer, thus defining a peculiar double positive distribution (Fig. 2). Similarly, the distribution of CD94 and CD159a antigens completely overlapped (data not shown). Moreover, CD11b and CD11c are expressed on subsets of CD18 positive cells (Fig. 2). On the other hand, CD56bright/CD18+ as well as CD94+ cells were CD117low while CD56bright/CD18− cells were CD117bright, suggesting that LFA-1 integrin, as CD94 and CD117 antigens (13, 14), might be a useful developmental marker, able to identify specific stages of CD56bright NK cells.
Identification of a Lineage Negative/CD34−/LFA-1low/CD161+/NKG2D+/NKp46+ Lymphocyte Subset Within PB and UCB Mononuclear Cells
To verify whether LFA-1 expression might be a marker of human NK development in vivo, we examined both in PB and UCB for discrete NK cell stages based on CD18-CD11a expression. Most of the analysis were performed on UCB, known to contain a higher percentage of NK cell progenitors compared to PB (3, 14). To focus on rare CD56bright NK cells, CD3, CD19, CD16, CD123, CD34 positive cells (i.e. T, B, CD56dim NK cells, basophils and hematopoietic progenitors) were gated out. The remaining lineage negative (Lin−)/CD34− lymphocytes were distinguishable in the well known populations: CD117bright/CD94− and its progeny, CD117low/−/CD94+ (13, 14 and data not shown). Interestingly, most of CD94bright cells were LFA-1bright (a typical CD56bright NK mature phenotype) while virtually all CD117bright cells expressed LFA-1 at low intensity (LFA-1low) (Fig. 3). This latter subset was detected within the lymphocyte region of UCB (0.35 ± 0.14%, mean ± S.D., n = 9) and PB samples (0.16 ± 0,10%, mean ± S.D., n = 5). Lin−/CD34−/LFA-1low showed significantly low forward and side scatter when compared with those of LFA-1bright cells, suggesting that the former are compatible with a small agranular morphology (Fig. 3). To focus on this immature subset, we performed a gate based on the scatter characteristics of LFA-1low cells (backgating using LFA-1low positive cells). Most of LFA-1low cells (>80%) expressed CD11b, CD7, and CD62L, and about 60–80% of them expressed CD161bright, NKG2Ddim, NKp46dim, CD244dim, and CD25bright (Fig. 3 and Table 3). Other antigens (CD94dim, NKp30dim, CD122dim, CD56dim, CD11cdim, CD2dim, CD117bright, CD33, and CCR7) were detected in lower percentages (<60%), while cell subsets expressing HLA-DR, CD8 and KIRs were mainly confined to “mature” Lin−/CD34−/LFA-1bright cells (Fig. 3 and Table 3). Within Lin−/CD34−/LFA-1low subset, contaminating CD5+ cells, presumably of non-NK origin, were usually around 10% (Table 3). As expected, the activation-induced markers NKp44 and TRAIL were expressed at low to undetectable percentages on both LFA-1low/ and LFA- 1bright/Lin−/CD34− cells (Fig. 3).
|Antigena||Percent within Lin−/CD34−/LFA-1low subset Mean SD (n)b|
|CD7||99 ± 1 (4)|
|CD62L||93 ± 3 (3)|
|CD11b||87 ± 5 (4)|
|CD161brightc||77 ± 7 (6)|
|CD25 bright||76 ± 4 (3)|
|NKG2Ddim||76 ± 7 (5)|
|NKp46dim||67 ± 7 (6)|
|CD244(2B4)dim||62 ± 6 (4)|
|CD117bright||42 ± 8 (6)|
|CD33||44 ± 18 (4)|
|CD122dim||49 ± 10 (4)|
|CD94dim||48 ± 15 (6)|
|CD2dim||45 ± 19 (6)|
|CD11cdim||44 ± 3 (5)|
|NKp30dim||40 ± 11 (5)|
|CD56dira||34 ± 17 (6)|
|CCR7||21 ± 10(3)|
|CD8dim||13 ± 13 (6)|
|CD5||10 ± 4 (6)|
Some of the most important information regarding human NK cell differentiation has been provided by multiparametric immunophenotyping of hematopoietic progenitor culture systems. This approach played a key role in the identification of novel stages of NK lineage and in the creation of NK development models. In a recent overview, Freud and Caligiuri (23) have proposed a sequential model of human NK differentiation, distinguishing five functionally different NK developmental stages (from NK progenitors to mature NK cells) based on surface antigen expression (CD34, CD117, CD94, and CD16).
In the present study, we characterized the CD56bright NK cells generated after 30 days of in vitro culture with an essential cytokine combination (FL plus IL-15). Interestingly, as shown in vivo by Shilling et al., this subset was the first to appear after about one month in hematopoietic stem cell transplants (24). However, the CD56bright cells obtained in culture have shown several immaturity traits as revealed by both the lack of β2-integrin and, characteristic of stage 3 NK cells (23), of CD94. As a matter of fact, low percentages of cells expressing LFA-1 or CD94 antigens have already been described in NK cells developing in vitro (10, 25). In our experiments, we have pointed out that, although the percentage of the CD94−/CD117bright NK subset (stage 3) resembled those of CD56bright/LFA-1− NK cells, a small proportion of CD56bright/LFA-1− cells expressed low density C-type lectin MHC-I inhibitory receptors, a reminiscence of the CD117low/CD94low intermediate population described by Grzywacz et al. (14). This indicates that in vitro LFA-1 integrin is upregulated slightly later than the CD94-CD159a heterodimer. On the basis of this observation, we hypothesized that LFA-1, as the β2 integrin CD11b for mouse NK cells (26), might be a useful marker to distinguish stages of human CD56bright NK cell development in vivo. To this regard, both UCB and PB samples contain a subset of Lin−/CD34− cells expressing LFA-1 at a lower density (LFA-1low) than the CD56bright “mature” cells. Of note, Lin−/CD34−/LFA-1low cells expressed some mature NK cell markers (i.e. CD94, CD11c, CD2, CD56, NKG2D, NKp46, NKp30, CD244, and CD122) at relatively low density, suggesting that they represent an immature stage of differentiation. In particular, a fraction of Lin−/CD34−/LFA-1low cells coexpressed CD56 and CD94 at low density, possibly defining an intermediate LFA- 1low/CD56dim/CD94dim/CD16− stage of the NK cell lineage. In agreement with this hypothesis, it has been recently described that CD56dim/CD16− NK cells can first develop into CD56bright/CD16− NK cells and then into CD56bright/CD16dim NK cells (27). Interestingly, like the perforin-deficient stage 3 human lymph node NK cells (13, 23), Lin−/CD34−LFA-1low are small agranular cells expressing CD7, CD161, CD62L, CD244, CD25, CD117bright, and CD33 antigens. However, differently from stage 3 cells, most of them expressed NKp46 and NKG2D molecules, resembling our in vitro findings. On the other hand, the deficiency of other activatory receptors, such as NKp44 and NKp30, is in line with the inducible nature of the former and the low expression density on CD56bright NK cells of the latter. In substance, most of the functional NK cell markers were similarly expressed in in vivo (PB and UCB) immature NK cells and in in vitro generated ones. This suggests that at least part of Lin−/CD34−/LFA-1low cells could represent the in vivo counterpart of CD161+/NCR+/LFA- 1−/CD94− NK cells, generated in vitro from CD34+ hematopoietic progenitor. Within Lin−/CD34−/LFA-1low cells, about half of them were CD161bright/CD56−/CD16−, an immature phenotype already described in PB and UCB (3) that has been shown to secrete IL-5 and IL-13 (12, 28). Interestingly, IL-13 has been described as being basically produced by CD56bright cells (5, 29). Finally, all these observations suggest that CD56bright NK cells may derive from CD161+/NKG2Ddim/NKp46dim/CD56− cells passing through the CD56dim/CD94dim/CD16− NK cell stage. Summarizing the reported data, we suggest the opportunity to distinguish two distinct maturative steps within the well known stage 3 of CD56bright differentiation, as described in Figure 4.
During normal in vivo differentiation, developing NK cells acquire activating and inhibitory receptors and cytotoxic function in a fashion that prevents NK-mediated auto-aggression against normal cells. There are different hypotheses to explain the NK cell self-tolerance during differentiation (20, 21). One possibility is that the expression of functional inhibitory receptors would precede that of activatory ones. Among human MHC-I inhibitory receptors, CD94-CD159a molecules have already been indicated as an early mechanism for self-tolerance during differentiation (20). Indeed, CD94-CD159a heterodimer is expressed earlier than KIRs and its inhibitory function has been demonstrated in NK cells differentiating in vitro (30, 31). However, our and other reports (4, 13) clearly indicate that activatory molecules are expressed in higher percentages of cells than MHC-I inhibitory receptors on immature NK cells, suggesting that activatory receptors precede CD94-CD159a heterodimer expression. In this regard, the inhibitory function of CD244 on the cytotoxic activity of immature NK cells has been claimed to assure a fine control of fail-safe mechanism (4). Nevertheless, the phenotype (LFA-1low) and the scatter (small agranular) characteristics of immature stage 3 NK cells can lead to further explanations. For example, they could not be still functionally cytotoxic due to the deficiency of β2-integrins and cytotoxic granules, and a complete functionality of NK cells could be reached only after the expression of MHC-I inhibitory receptors.
Concluding, although further investigations are needed, we propose LFA-1 as a useful marker to identify immature stages of NK cell differentiation.
The authors are grateful to Drs. Mario Buonvino, Claudia Masoni, and Massimo Della Felice, for technical assistance; Drs. Giuliana Gobbi and Emanuela Marcenaro for critically reading the manuscript. Thanks to Dr. Eugenio Fusco for providing cord blood samples, Dr. Franco Monti for AB serum, Prof. Alessandro Moretta for anti-CD244 (clone CO54) mAb, Instrumentation Laboratory S.p.A., Space Import Export S.r.l. and Dr. Valter Occhiena for various reagents.