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CD56^brightCD16^– Killer Ig-Like Receptor^– NK Cells Display Longer Telomeres and Acquire Features of CD56^dim NK Cells upon Activation¹

Chiara Romagnani^2,*, Kerstin Juelke^*, Michela Falco, Barbara Morandi, Antonella D’Agostino, Roberta Costa, Giovanni Ratto^¶, Giuseppe Forte^||, Paolo Carrega^#, Gabrielle Lui, Romana Conte^¶, Till Strowig^**, Alessandro Moretta^#,, Christian Münz^**, Andreas Thiel^*, Lorenzo Moretta^,#, and Guido Ferlazzo^2,,**

^* Department of Clinical Immunology, German Rheumatism Research Centre, Deutsches Rheuma-Forschungszentrum Berlin, Berlin, Germany; Istituto Giannina Gaslini, Largo Gaslini, Genoa, Italy; Department of Human Pathology, School of Medicine, University of Messina, Messina, Italy; Ospedale Santi Antonio Biagio Cesare Arrigo, Alessandria, Italy; ^¶ Istituto Nazionale Ricerca sul Cancro, Genoa, Italy; ^|| Ospedale Santa Croce e Carle, Cuneo, Italy; ^# Department of Experimental Medicine, University of Genova, Genova, Italy; ^** Laboratory of Viral Immunobiology, and Christopher H. Browne Center for Immunology and Immune Diseases, The Rockefeller University, New York, NY 10021; and Centro di Eccellenza Ricerca Biomedica, University of Genova, Genova, Italy

	Abstract

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Human NK cells can be divided into CD56^dimCD16⁺ killer Ig-like receptors (KIR)^+/– and CD56^brightCD16^– KIR^– subsets that have been characterized extensively regarding their different functions, phenotype, and tissue localization. Nonetheless, the developmental relationship between these two NK cell subsets remains controversial. We report that, upon cytokine activation, peripheral blood (PB)-CD56^bright NK cells mainly gain the signature of CD56^dim NK cells. Remarkably, KIR can be induced not only on CD56^bright, but also on CD56^dim KIR^– NK cells, and their expression correlates with lower proliferative response. In addition, we demonstrate for the first time that PB-CD56^dim display shorter telomeres than PB- and lymph node (LN)-derived CD56^bright NK cells. Along this line, although human NK cells collected from nonreactive LN display almost no KIR and CD16 expression, NK cells derived from highly reactive LN, efferent lymph, and PB express significant amounts of KIR and CD16, implying that CD56^bright NK cells could acquire these molecules in the LN during inflammation and then circulate through the efferent lymph into PB as KIR⁺CD16⁺ NK cells. Altogether, our results suggest that CD56^brightCD16^– KIR^– and CD56^dimCD16⁺KIR^+/– NK cells correspond to sequential steps of differentiation and support the hypothesis that secondary lymphoid organs can be sites of NK cell final maturation and self-tolerance acquisition during immune reaction.

	Introduction

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For many years, NK cells have been regarded as a homogeneous lymphocyte population characterized by strong cytotoxic capability. More recently, NK cells have been shown to comprise various subsets that differ in function, phenotype, and tissue localization. The majority of peripheral blood (PB)³-NK cells (95%) belong to the CD56^dimCD16⁺ cytolytic NK subset (1, 2) and express homing receptors for inflamed peripheral sites and lytic granules to rapidly mediate cytotoxicity (1, 3). The remaining PB-NK cells (5%) are represented by CD56^brightCD16^– cells (2), which conversely express very low levels of lytic granules, secrete larger amounts of IFN- and TNF, and proliferate much more vigorously than CD56^dimCD16⁺ NK cells upon activation (4). Phenotypically, CD56^brightCD16^– NK cells, unlike CD56^dimCD16⁺, express the receptor for stem cell factor (c-kit or CD117), the -chain of IL-7R, and secondary lymphoid organ (SLO) homing markers, namely CCR7, CD62L, and CXCR3 (2, 4, 5). Notably, the MHC class I allele-specific killer Ig-like receptors (KIR) are expressed on a considerable fraction of CD56^dimCD16⁺ NK cells, whereas the CD56^brightCD16^– NK subset lacks KIR (1). Recent reports have shown that a substantial amount of human NK cells resides in SLO, representing up to 5% of mononuclear cells in noninflamed lymph nodes (LN) (6, 7). As it might be predicted from their homing receptor expression, CD56^bright NK cells are enriched in all SLO analyzed to date (LN, tonsils, and spleen) (7). Like PB-CD56^bright NK cells, SLO-NK cells exhibit no KIR or CD16 expression and poor cytolytic activity. However, SLO-NK cells promptly acquire cytotoxic ability and the expression of most inhibitory and activating receptors upon IL-2 stimulation (7). Therefore, activation converts SLO-NK cells into cytotoxic effectors analogous to blood CD56^dimCD16⁺ NK cells. Whether upon activation PB-CD56^bright NK cells also may convert into KIR⁺ CD16⁺ NK cells remains to date to be established. In this regard, controversial hypotheses have been proposed recently for the developmental relationship between these two NK cell subsets because CD56^bright KIR^– CD16^– NK cells have been suggested either to represent precursors of CD56^dim KIR^+/– CD16⁺ cells or to be derived from CD56^dim KIR^+/– CD16⁺ cells (8, 9, 10). Studies addressing this question have been hampered by the lack of CD56 in mice and by missing information regarding the site of terminal NK cell differentiation.

In our present study, we investigate whether PB-CD56^bright NK cells give rise in vitro and in vivo to cells akin of CD56^dim NK cells and whether SLO can be sites of NK cell maturation. All of the observations collected along our present study support the hypothesis that CD56^dim cells derive from the CD56^bright NK cell subset and that this differentiation can take place during immune activation in inflamed peripheral tissues, such as reactive LN.

	Materials and Methods

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Cell preparations and cultures

Whole blood and leukocyte concentrates were collected from healthy donors. LN, thoracic duct lymph, and blood were obtained from patients who underwent surgery for cancer resection. All sample collections were obtained after donor informed consent and approval by our Institutional Ethics Committee. When histological evaluation of LN was required, LN were incised immediately after removal and cut into two parts, one of which was paraffin embedded to perform histology, whereas the other was processed for single-cell isolation. For isolation of LN single cells, LN were mechanically dissociated and then treated with enzymes, as previously described (7). NK cells were enriched from PB and LN by negative selection using NK Cell Isolation Kit II (Miltenyi Biotec). To obtain highly purified CD56^bright and CD56^dim NK cell subsets, cells were FACS sorted directly or after MACS-negative selection using FACSAria (BD Biosciences), according to the lack of CD3 and the expression of CD56 and additional markers, when indicated in the text. For isolation of naive and memory CD4⁺ T cells, CD4⁺ T cells were first positively selected using CD4 Microbeads (Miltenyi Biotec) and then FACS sorted after staining with anti-CD4, anti-CD45RA, anti-CD45RO, and anti-CD27 mAbs. All sorted subsets used for the experiments always displayed purity above 97%. The medium used throughout experiments was RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FCS (Invitrogen Life Technologies) and 1% antibiotic mixture (5 mg/ml penicillin, 5 mg/ml streptomycin stock solution). Unless differently indicated in the text, 10⁵ NK cells were cultured in 96-well round-bottom plates (Costar) in the presence or absence of the following cytokines: 100 IU/ml IL-2 (Roche), 25 ng/ml IL-12, or 25 ng/ml IL-15 (R&D Systems). To evaluate proliferation, cells were labeled with CFSE (Molecular Probes), as previously described (7).

Flow cytometric phenotypic analysis

The following Abs, anti-CD56 PE Cy7 (NCAM16.2), anti-CD117 allophycocyanin (YB5.B8), anti-CD16 PE or allophycocyanin Cy7 (clone 3G8), anti-CD62L PE or allophycocyanin (Dreg56), anti-CXCR3 allophycocyanin (1C6/CXCR3), anti-granzyme A PE (CB9), anti-CD45RA PE Cy7, and anti-CD45RO FITC, were purchased from BD Pharmingen. Anti-granzyme B PE (GB12) was purchased from Caltag Laboratories. Anti-CD56 PE or allophycocyanin (AF12-7H3) was purchased from Miltenyi Biotec. Anti-CD127 PE (R34.34) was purchased from Beckman Coulter. Anti-KIR3DL1 PE (DX9) was purchased from Abcam. For CCR7 staining, cells were first incubated with purified anti-CCR7 mAb (IgG2a, clone 150503; R&D Systems) and afterward with a biotin-conjugated mAb directed against IgG2a (Southern Biotechnology Associates). Anti-CD3 (OKT3), anti-CD4 (TT1), and anti-CD27 (2E4) mAbs were produced and conjugated in our laboratory either with FITC, PE, or Cy5. Anti-KIR2DL2/L3/S2 (GL183), anti-KIR2DL1/S1 (EB6), and anti-KIR3DL1/L2/S1 (Az158) mAbs were also produced and conjugated in our laboratory either with FITC, Cy5, or biotin. When cells were stained with biotin-conjugated mAbs, streptavidin-Pacific Blue or streptavidin-Alexa647 (Molecular Probes) were used as secondary reagents. For phenotypic analysis, data were acquired on a FACSCalibur or LSRII flow cytometer, the latter using Diva Software 3.0 and 4.1.2 (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star).

RT-PCR analysis of KIR transcripts

Total RNA was extracted from CD56^bright, CD56^dim KIR^–, and CD56^dim KIR⁺ NK cells at day 0 directly after sorting or after a 5-day culture in the presence of 100 IU/ml IL-2 using RNeasy micro kit (Qiagen), according to manufacturer’s instruction. cDNA synthesis was performed on 500 ng of RNA using oligo(dT) primers. Three different sets of primers were used in this study. KIR up: CAT GTY GCT CAY KGT CGT C and KIR down: GGT TTT GAG ACA GGG CTG allowed the amplification of the KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1 open reading frame (ORF). The sets of primers common up/C and common up/E allowed the amplification of a segment of activating and inhibitory KIR transcripts, respectively, as previously described (11). The PCR products were resolved into 0.8% agarose gel.

Analysis of telomere length and telomerase activity

Analysis of telomere length was performed using a quantitative flow-fluorescence in situ hybridization (FISH) methodology that employs a fluorescein-conjugated peptide nucleic acid (PNA) probe (Telomere PNA Kit/FITC for Flow Cytometry; DakoCytomation), according to manufacturer instructions. Briefly, a single-cell suspension (either PB-CD56^dim, PB-CD56^bright, LN-NK cells, naive, or memory CD4⁺ T cells) was obtained and mixed with control cells (i.e., the 1301 cell line), which display very long telomeres. Mixed cell suspension DNA was denatured for 10 min at 82°C either in hybridization solution without probe or in hybridization solution containing fluorescein-conjugated PNA telomere probe. Hybridization took place in the dark at room temperature overnight and was followed by two washes at 40°C. After propidium iodide staining, flow cytometric analysis was performed gating on G_0/1 cells. The relative telomere length (RTL) value was calculated as the ratio between the telomere signal of each sample and the control cells (1301 cell line) with correction for the DNA index of G_0/1 cells. This correction was performed to standardize the number of telomere ends per cell and thereby telomere length per chromosome.

Quantitative determination of telomerase activity was performed on highly purified CD56^bright and CD56^dim NK cells by a photometric enzyme immunoassay (Telo TAGGG Telomerase PCR ELISA^plus; Roche), according to the manufacturer instructions.

Statistical analysis

Statistics were calculated using Student’s t test or Mann-Whitney U test.

	Results

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PB-CD56^bright NK cells can express KIR and CD16 upon cytokine stimulation

Some reports have shown that CD16 can be down-regulated on PB-CD56^dim NK cells (9, 10), suggesting that CD56^bright NK cells might represent activated CD56^dim NK cells. Nevertheless, down-regulation of KIR expression from PB-CD56^dim KIR⁺ NK cells has never been reported to date. To identify variations of KIR expression on the distinct PB-NK cell subsets, NK cells were sorted with high purity after staining with anti-CD56, anti-CD3, and a combination of anti-KIR (anti-KIR2DL2/L3/S2, anti-KIR2DL1/S1, anti-KIR3DL1/L2/S1) Abs. CFSE-labeled CD56^bright (excluding the few KIR⁺ events), CD56^dim KIR⁺, and CD56^dim KIR^– NK cells were cultured in the presence of IL-2, IL-12, or IL-15 and analyzed for KIR surface expression. As shown in Fig. 1A, both CD56^bright KIR^– (top row) and CD56^dim KIR^– NK cells (middle row) exhibited de novo expression of KIR on a significant proportion of cells in response to IL-2 (and IL-15; data not shown) and in lower proportion in response to IL-12. In contrast, none of these stimuli was able to down-regulate KIR expression on sorted CD56^dim KIR⁺ NK cells, which was conversely up-regulated after IL-2 stimulation (Fig. 1A, bottom row). As previously shown, CD56^bright NK cells displayed a higher proliferative response to IL-2, IL-15, and IL-12 compared with total CD56^dim NK cells. Nonetheless, the comparison of the proliferative ability at day 5 of CD56^bright in response to 100 IU/ml IL-2 (percentage of mean proliferation ± SEM = 92.5 ± 1.6%) with CD56^dim KIR^– (67.7 ± 9.5%) or KIR⁺ NK cells (26.4 ± 5.8%) showed that CD56^bright proliferate slightly more than CD56^dim KIR^– NK cells (p 0.05), whereas CD56^dim KIR⁺ proliferate significantly less compared not only to CD56^bright (p 0.002), but also to KIR^– NK cells (p 0.015) (Fig. 1A), suggesting that expression of KIR might correlate with a terminally differentiated phenotype. Notably, the lower proliferative capacity of KIR⁺ NK cells excludes the possibility that rare contaminating KIR⁺ NK cells could overgrow CD56^bright and CD56^dim KIR^– NK cells and be responsible for KIR expression among KIR^– NK cells.

View larger version (62K): [in this window] [in a new window]   FIGURE 1. PB-CD56bright and CD56dim KIR– NK cells express KIR upon cytokine activation. Sorted NK cell subsets were CFSE labeled and cultured in the presence or absence of IL-2 or IL-12. At day 5, modulation of KIR molecules was evaluated after staining with different mAbs or at the mRNA level. Percentages of KIR+ cells are indicated in each dot plot. A, CFSE-labeled CD56bright, CD56dim KIR–, and CD56dim KIR+ NK cells were stained with a combination of Abs specific for KIR2DL2/L3/S2, KIR2DL1/S1, and KIR3DL1/L2/S1. One representative experiment of six is shown. B, Total NK cells were stained to check ex vivo expression of single KIR after staining with Abs directed either against KIR2DL2/L3/S2, KIR2DL1/S1, or KIR3DL1 together with anti-CD56 mAb (left column). CD56bright, CD56dim KIR–, and CD56dim KIR+ NK cells were then sorted after staining with a combination of Abs specific for all KIR (KIR2DL2/L3/S2, KIR2DL1/S1, and KIR3DL1/L2/S1) together with anti-CD56 mAb. Purity check of the three sorted subsets from a representative experiment is shown (middle column). After 5-day culture in the presence of IL-2, up-regulation of each single KIR was checked on the three sorted NK cell subsets after staining with Abs directed either against KIR2DL2/L3/S2, KIR2DL1/S1, or KIR3DL1 together with anti-CD56 mAb (right columns). One representative experiment of three is shown. C, RT-PCR analysis of total RNA isolated from sorted CD56bright, CD56dim KIR–, and CD56dim KIR+ NK cell subsets derived from the same donor shown in B. PCR products obtained using sets of primers specific for KIR3D (first lane, higher band) and KIR2D (first lane, lower band) ORF transcripts (see Materials and Methods), and their inhibitory (second lane) or activating (third lane) counterparts were resolved into 0.8% agarose gel. RT-PCR of a -actin segment (fourth lane) was used as positive transcription control. Analysis was performed directly after sorting or after a 5-day culture in the presence of IL-2. One representative experiment of two is shown.

To investigate modulation of CD16 expression, NK cells were sorted after staining with anti-CD56, anti-CD3, and anti-CD16 Abs. CFSE-labeled CD56^brightCD16^– and CD56^dimCD16⁺ NK cells were cultured, as described in Fig. 1A, and analyzed for CD16 surface expression. Fig. 2 shows that significant CD16 up-regulation occurs on the majority of CD56^brightCD16^– NK cells after IL-2 (and IL-15; data not shown) stimulation, whereas, as for KIR induction, IL-12 was less efficient. Partial down-regulation of CD16 expression occurs on CD56^dimCD16⁺ NK cells in the presence of IL-12, as previously reported (9), as well as when cells were left for 5 days in medium alone, although they have been sorted previously with very high purity for CD16 expression (Fig. 2). Stimulation with IL-18 alone or in combination with IL-2, which can down-regulate CD16 expression on CD56^dim NK cells (10), was conversely not able to modulate KIR expression on either CD56^bright or CD56^dim NK cells (data not shown).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. PB-CD56bright NK cells up-regulate CD16 expression upon cytokine activation. CD56brightCD16– and CD56dimCD16+ NK cells were sorted, CFSE labeled, and cultured in the presence or absence of IL-2 or IL-12. At day 5, modulation of surface CD16 expression was evaluated after staining with anti-CD16 Ab. Percentages of CD16+ cells are indicated in each dot plot. One representative experiment of five is shown.

CD56^bright NK cells acquire signature of CD56^dim NK cells upon cytokine activation

In addition to CD16 and KIR, many other molecules have been described to be differentially expressed in CD56^bright and CD56^dim NK cells. Although analysis of CD56^bright vs CD56^dim vs IL-2-activated CD56^dim NK cell gene signature has been performed (5), no extensive analysis exists concerning modulation of PB-CD56^bright NK cell phenotype, in particular during proliferation. To test the hypothesis that, upon cytokine activation, CD56^bright can acquire the signature of CD56^dim NK cells, CFSE-labeled PB-CD56^bright and CD56^dim NK cells were cultured with IL-2, IL-12, or IL-15 for 5 days and then analyzed for the expression of markers that are mutually expressed on one of the two subsets, i.e., IL-7R, c-kit, CXCR3, CCR7, CD62L (mainly expressed on CD56^bright NK cells), granzyme A, and granzyme B (almost confined to CD56^dim NK cells). Modulation of the expression of each molecule was evaluated comparing its mean fluorescence intensity (MFI) among cells that have undergone extensive proliferation after 5-day cytokine stimulation with the MFI among resting cells cultured in medium (Fig. 3, A and B, first column) for the same time period. Notably, expression of each molecule analyzed ex vivo did not significantly change after 5-day culture in medium alone, i.e., without cytokines (data not shown). As shown in Fig. 3A, CD56^bright NK cells were able to down-regulate surface expression of IL-7R, c-kit, CXCR3, and CCR7 after IL-2 and IL-12 (and IL-15; data not shown) stimulation in proliferating cells compared with resting cells kept in medium for the same time interval. Notably, down-regulation of cell surface expression of these markers is not necessarily related to proliferation, because MFI decreased already in nondividing cells cultured in the presence of cytokines when compared with the ones kept in medium only. CD62L expression on CD56^bright NK cells was strongly down-regulated after IL-2 (and IL-15; data not shown) activation, although even up-regulated by IL-12 independently of proliferation. While entering the cell cycle, CD56^dim NK cells did not acquire any of these molecules under the same culture conditions, with the exception of CD62L, which was partly up-regulated on CD56^dim NK cells after IL-12 stimulation (Fig. 3B). Intracellular staining of CFSE-labeled CD56^bright NK cells after IL-2, IL-15, or IL-12 stimulation revealed that cytolytic molecules such as granzyme A and granzyme B were acquired by CD56^bright NK cells upon all stimulating conditions (Fig. 3A, and data not shown for IL-15), whereas the same molecules remained rather stable in CD56^dim NK cells (Fig. 3B). Stimulation with IL-18 alone or in combination with IL-2 was conversely not able to significantly modulate the expression of any of the markers analyzed, neither on CD56^bright or CD56^dim NK cells, apart from CCR7, which, as previously described (10), was up-regulated on CD56^dim NK cells. Notably, CD83 which has also been shown to be up-regulated on NK cells after IL-18 stimulation (10), could be induced not only on CD56^dim, but also on CD56^bright NK cells (data not shown).

View larger version (75K): [in this window] [in a new window]   FIGURE 3. PB-CD56bright NK cells acquire signature of CD56dim NK cells upon cytokine activation. Sorted CD56bright (A) or CD56dim NK cells (B) were CFSE labeled and cultured in the presence of medium, IL-2, or IL-12. Because a small fraction of CD56dim NK cells can express CD62L, for this experiment (fifth row), CD56dim NK cells were negatively sorted for CD62L. At day 5, cells were stained with their correspondent mAbs, and modulation of the indicated surface or intracellular molecule was evaluated comparing MFI among resting (right gate) and proliferating (left gate) cells, as depicted in each dot plot. Slight differences in CFSE intensity and in the proliferative ability of CD56bright (mean percentage of proliferation ± SEM = 90.7 ± 3.6 for IL-2 and 53.4 ± 7.1 for IL-12) or CD56dim NK cells (mean percentage of proliferation ± SEM = 66.6 ± 7.2 for IL-2 and 34.1 ± 7.3 for IL-12) are due to the fact that a distinct donor was used for each marker. To avoid flow cytometer compensation inconveniences due to counterstaining with CFSE, fluorescence intensities and compensation were set based on isotype controls, in which MFI in proliferating and resting cells remains constant. Notably, due to the use of two versions of Diva Software (3.0 and 4.1.2) on LSRII, dot plots with two different logarithmic scales are shown. For each molecule analyzed, one representative experiment of three is shown.

CD56^bright NK cells derived from PB and LN display longer telomeres than PB-CD56^dim NK cells

To address whether CD56^bright NK cells represent an earlier differentiation step of CD56^dim NK cells, we evaluated telomere length in NK cell subsets isolated ex vivo from PB and LN. The measurement of telomere length has been widely used for assessing the proliferative history of distinct cell subsets, among which are naive and memory T cells (12, 13, 14, 15, 16, 17). In most normal somatic cells, telomere sequences are lost during replication, and therefore telomere length inversely correlates with cell age. In our study, we used flow-FISH because this technique allows the assessment of telomere length with greater sensitivity than traditional methodologies (14, 15).

As shown in Fig. 4, in seven donors analyzed, sorted PB-CD56^dim NK cells displayed significantly shorter telomere length than autologous PB-CD56^bright NK cells (p 0.01), with a mean telomere shortening of 15.3% in the CD56^dim compared with the CD56^bright NK cells (Fig. 4, B and C). As a comparison, we assessed telomere length difference also in naive CD45RA⁺CD45RO^–CD27⁺ and memory CD45RA^–CD45RO⁺CD4⁺ T cells derived from PB of two donors analyzed in Fig. 4, B and C, for NK cell subsets. Fig. 4D shows that telomere shortening in memory compared with naive CD4⁺ T cells matches up with the one observed in CD56^dim compared with CD56^bright NK cells (24.3 and 15.4%, respectively). Notably, CD56^bright NK cells displayed RTL similar to naive T cells, whereas CD56^dim NK cells to memory T cells. In addition, comparative analysis of telomere length in PB-CD56^dim NK cells and autologous LN-NK cells, which are predominantly CD56^bright, revealed that PB-CD56^dim NK cells significantly displayed shorter telomeres than LN-NK cells (p 0.04), with a mean telomere shortening of 14.5% in the PB-CD56^dim compared with the LN-CD56^bright NK cells (Fig. 4, E and F). A similar degree of telomere cutback was observed when LN-NK cells were sorted and cultured for as long as 3 wk in the presence of 100 IU/ml IL-2 (Fig. 4G).

View larger version (31K): [in this window] [in a new window]   FIGURE 4. PB-CD56bright and LN-NK cells display longer telomeres than PB-CD56dim. PB-CD56bright, PB-CD56dim, and LN-NK cells were sorted with high purity from different donors (A, one representative PB-NK cell sorting is depicted) and evaluated for their telomere length by flow-FISH (each experiment shown was performed in duplicate). B, RTL of PB-CD56bright and PB-CD56dim NK cells are shown. RTL was calculated as the ratio between the fluorescence intensities of each sample and control cells (1301 cell line). Each bar represents the mean RTL value ± SD of CD56bright NK cells () compared with the CD56dim NK cells () in seven individual donors (**, p 0.01, as calculated by paired Student’s t test). C, RTL values between PB-CD56dim () and PB-CD56bright NK cells () were also compared by setting the latter ones as 100% and shown for all seven donors. D, RTL values of PB-CD56bright () and CD56dim NK cells () derived from two donors, included in B and C, were compared with RTL values of autologous CD45RA+ RO– CD27+ naive (black striped bar) and CD45RO+ RA– memory (gray striped bar) CD4+ T cells. Each bar represents the mean RTL value ± SD. E, Telomere length of LN-NK cells () and PB CD56dim NK cells () was compared in four donors. Each bar represents the mean RTL value ± SD (*, p 0.04 by paired Student’s t test). F, RTL values between PB-CD56dim () and autologous LN-NK cells () were also compared after setting the latter ones as 100% and shown for all four donors. G, Telomere length was analyzed in total LN-derived NK cells sorted ex vivo (RTL set as 100%, ), in the same cells after 3-wk culture with 100 IU/ml IL-2 () and in autologous PB-CD56dim NK cells (). Each bar represents the mean RTL value ± SEM.

These results indicated that CD56^bright NK cells have undergone a considerable smaller number of cell divisions in vivo as compared with CD56^dim and might therefore represent an upstream developmental stage of NK cells.

NK cells isolated from efferent lymph, but not LN-NK cells, express CD16 and KIR

It has been proposed recently that SLO might represent sites of differentiation for NK cells (7, 8), which would eventually colonize blood and other peripheral tissues. We hypothesized that if SLO were indeed sites of NK cell differentiation, NK cells leaving LN should be different from NK cells resident in LN. To this aim, we analyzed in parallel NK cells isolated from the efferent lymph system (i.e., thoracic duct) and from autologous LN. Consistent with previous reports (6, 7), NK cells harbored in nonreactive LN displayed low or no KIR and CD16 expression. In contrast, a significant proportion of NK cells collected from the efferent lymph of the thoracic duct expressed KIR and CD16, although the latter to a lower extent than their blood counterpart (Fig. 5A). CD56 expression was still bright as compared with PB, suggesting that this marker might be down-regulated in vivo at later time points, as also indicated by our results obtained in vitro. These data cannot rule out the possibility that, conversely, a small percentage of CD56^dim NK cells expressing KIR and CD16 enters the LN, expands in situ, and then leaves LN via efferent lymph. Nonetheless, considering the lower proliferative ability and chemokine receptor expression of CD56^dimCD16⁺KIR⁺ NK cells, we favor the hypothesis that NK cells can acquire de novo expression of relevant functional molecules in LN and then circulate to PB through the efferent lymph.

View larger version (50K): [in this window] [in a new window]   FIGURE 5. NK cells from efferent lymph and from LN with paracortical/follicular hyperplasia express KIR and CD16. A, Mononuclear cells were isolated from efferent lymph (i.e., thoracic duct), autologous LN, and PB. CD16 and KIR expression was analyzed gating on CD56+CD3– NK cells. One representative experiment of two is shown. B, Sections of paraffin-embedded tissue of 28 LN were stained with H&E. Three LN representative of distinct inflammatory conditions detected by the histological analysis of the 28 analyzed are shown. i, Nonreactive LN, magnification x5. ii, Detail of a follicle shown in i, magnification x40. iii, LN paracortical hyperplasia: manifest enlargement of the paracortical area between capsula and follicles, magnification x5. iv, Detail of the paracortical area shown in iii, magnification x40. v, LN follicular hyperplasia, magnification x5. vi, Detail of the follicular hyperplasia showing the presence of several plasma cells. C, Percentage of KIR-expressing NK cells isolated from LN exhibiting paracortical or follicular hyperplasia or combination of the two (triangles) or from LN displaying no reactivity or sinus hyperplasia (circles), according to the histological analysis. The median value for each group is also depicted (p 0.0018 by Mann-Whitney U unpaired test). To correlate LN inflammatory status and NK cell KIR or CD16 expression, LN were incised immediately after removal and split into two sagittal parts, one of which was paraffin embedded to perform histology, whereas the other was processed for single-cell isolation and analyzed by flow cytometry.

The latter observation that NK cells emigrating from, but not resident in LN, also express KIR and CD16 (Fig. 5A) prompted us to speculate that only reactive LN might be site of NK cell activation and KIR and CD16 acquisition. We therefore analyzed NK cells isolated from 28 LN of 14 individual donors with the aim of investigating whether KIR and CD16 expression might be present in reactive LN. Remarkably, a significant percentage of NK cells expressing KIR was detectable only in LN characterized by paracortical/follicular hyperplasia (mean percentage ± SEM: 7.3 ± 0.3; Fig. 5C), which is characterized by the presence of secondary follicules and lymphocyte proliferation (Fig. 5B, iii–vi). NK cells isolated from nonreactive LN (Fig. 5B, i and ii) or LN with sinus hyperplasia (characterized by an increased number of macrophages) showed low or no expression of KIR (mean percentage ± SEM: 1.75 ± 0.2; Fig. 5C). Similarly to KIR, CD16 expression also correlated with LN-paracortical/follicular hyperplasia (data not shown). Because of the striking association of KIR and CD16 expression with LN-paracortical/follicular hyperplasia (p 0.002) and according to previous reports demonstrating that PB-NK cells can reach, but promptly leave, inflamed LN within 72 h (18), we are tempted to speculate that KIR expression in LN NK cells might represent a de novo induction of these molecules occurring on LN resident CD56^bright NK cells in the course of an inflammatory immune response, characterized by the abundant presence of different cytokines (e.g., IL-15, IL-12, and IL-2).

	Discussion

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Despite progress in understanding NK cell specificity for target cells, less is known about stages of NK cell maturation, expansion, and site of differentiation, especially in the human system. This study sheds some light on the final stages of human NK cell differentiation, showing that after cytokine activation, in particular IL-2 and IL-15, CD56^bright NK cells could acquire the signature of CD56^dim NK cells, i.e., KIR⁺ CD16⁺ IL-7R^– c-kit^– CXCR3^– CCR7^–, CD62L^–, whereas CD56^dimCD16⁺ KIR⁺ NK cells substantially maintain their features of terminally differentiated cells. Within the molecules acquired by CD56^bright NK cells, KIR are of great interest: in contrast to CD16 expression, which has been shown to be differentially modulated in several experimental models (9, 10), once a NK cell has acquired its specific set of KIR, the expression remains stable, as shown in NK cell clones under varying cell culture conditions and activation stimuli (19). KIR expression is actually regulated by epigenetic mechanisms, and it had been shown to date that it could be acquired in vitro only using methyltransferase inhibitors (19). Indeed, our data demonstrate that both CD56^bright KIR^– and CD56^dim KIR^– NK cells could acquire KIR expression on a subset of cells after cytokine stimulation. This result is apparently in contrast to a previous publication in which it was claimed that no KIR or CD16 up-regulation was occurring on CD56^bright NK cells after IL-2 stimulation (1). One possible explanation for this discrepancy might rely on the different experimental conditions (e.g., IL-2 concentration). Conversely, the time frame analyzed is comparable and, by performing a kinetic of KIR expression in CD56^bright NK cells stimulated with IL-2 up to 40 days after sorting, we could observe that the percentage of up-regulated KIR remains constant (data not shown).

The evidence that KIR are molecules inducible on NK cells raises interest about the mechanisms by which this process can occur, because this understanding might help to clarify the mechanisms underlying NK cell tolerance to self. Recent studies have changed our view on how NK cell self-tolerance is achieved, showing that NK cells, which do not express inhibitory receptors recognizing self-MHC, do exist. Nonetheless, only NK cells expressing inhibitory receptors that recognize self-MHC are competent, whereas those that do not display an anergic phenotype (20, 21, 22, 23, 24). In this context, our finding that KIR can be induced by cytokines is of great interest because it suggests that NK cell self- tolerance might be a dynamic process probably related to cell differentiation: cytokines produced during an inflammatory response by dendritic cells (DCs) or T cells would induce NK cell differentiation and generation of new competent NK cells. Along this line, it has been shown also that Ly49 molecules can be modulated on NK cells after cytokine stimulation, suggesting a similar scenario in mice (25). Our findings about KIR as inducible receptors might also have relevant implications for manipulating NK cell self-tolerance in clinical settings such as organ transplantation, in which KIR specificity has already been shown to be relevant for tumor rejection (26).

KIR⁺ NK cells showed lower ability to proliferate in response to cytokines compared not only to CD56^bright, but also to CD56^dim KIR^–. This surprising finding was not due to a mAb-staining artifact (e.g., inhibition of proliferation via triggering of inhibitory receptors by anti-KIR mAb) because staining or not total CD56^dim NK cells (mixture of KIR⁺ and KIR^– NK cells) with anti-KIR mAb combination used for our sorting procedure did not influence at all NK cell proliferation (data not shown). One possible explanation of the lower CD56^dim KIR⁺ NK cell proliferation might be due to KIR binding in cis or in trans to surface MHC class I molecules expressed on NK cells, which could result in inhibition of proliferation after stimulation, as it has been shown for cis binding of Ly49 in mice (27). Nonetheless, we tend to exclude this hypothesis because, also in this case, staining of KIR with mAb should influence KIR ability to bind MHC class I molecules and resulting in less inhibition. Given all these observations, we favor the hypothesis that CD56^bright and CD56^dim NK cells do not belong to two distinct subsets, each one displaying dissimilar proliferative features, but rather that CD56^bright represent an earlier stage of NK cell development and that KIR acquisition correlates with a terminal step of NK cell maturation, as it has been speculated already both for NK cells and CD8⁺ KIR⁺ T cells (28, 29). CD56^bright NK cells also down-regulated the expression of cytokine receptors such as the stem cell factor receptor CD117 (c-kit) and CD127 (IL-7R) during cytokine-induced proliferation. The progressive loss of receptors that are selectively expressed not only by CD56^bright, but also by NK cell immature precursors in humans and in mice (30, 31), is also very suggestive of a differentiation process from an early to a more advanced stage at which NK cells do not require stem cell factor or IL-7 signaling any longer. Accordingly, mouse Mac^low NK cells displaying an immature phenotype tend to express c-kit, which is then absent in mature Mac^high NK cells (31). Down-regulation of SLO homing molecules such as CD62L, CXCR3, and CCR7 on CD56^bright NK cells upon cytokine stimulation is consistent with the hypothesis that these NK cells would leave SLO after their activation. If CD56^bright NK cells represented an earlier developmental step of NK cell differentiation, they should have undergone a lower number of proliferative events in vivo. Consistent with this hypothesis, CD56^bright NK cells display longer telomeres than CD56^dim NK cells. In this regard, we could demonstrate that CD56^bright exhibit the same characteristics of naive T cells, i.e., longer telomeres compared with memory T cells, according to previous reports (17). Although not providing the definitive proof that CD56^bright are the precursors of CD56^dim NK cells, these findings definitely rule out the hypothesis that CD56^bright are derived from CD56^dim NK cells and strengthen their close functional and molecular resemblance with naive T cells.

Ex vivo analysis of human NK cells from different compartments revealed that nonreactive LN contain almost exclusively CD56^bright KIR^– CD16^– NK cells, whereas a significant NK cell expression of KIR and CD16 is present in highly inflamed LN and in the efferent lymph. These data suggest that CD56^bright KIR^– CD16^– NK cells can acquire KIR and CD16 in inflamed LN and then circulate via the efferent lymph in PB as KIR⁺ CD16⁺ NK cells. The assumption that cytokines released during inflammation can mobilize NK cells from SLO to PB is also supported by previous studies reporting that rIL-2 therapy for human cancer results in a striking increase of CD56^brightCD16⁺ NK cells in PB (32). Because these NK cells are not cycling (33), it is conceivable that they are mobilized from extravascular tissues, rather than directly proliferating in the blood. It could be envisaged that in steady state or very early during an immune response, CD56^bright KIR^– NK cells can be recruited into LN (18, 34), whereas later on during inflammation mature NK cells leave LN and then circulate in PB to reach inflamed tissues. Although this hypothesis is very challenging, we cannot exclude that the presence of KIR⁺CD16⁺ NK cells in inflamed LN (and in the efferent lymph) might be due to selective migration of this subset into LN and not to CD56^bright differentiation into CD56^dim NK cells. Nonetheless, because it has been shown that NK cell recruitment into inflamed LN occurs via CD62L and CXCR3 (18), KIR⁺CD16⁺ NK cells, which are generally CD62L^– and CXCR3^–, should be less prone to migrate to this site.

Other studies have also suggested that LN may represent a key site for NK cell development (8, 30). In fact, it has been shown recently that four different developmental stages of human NK cell precursors are present in LN and that differentiation from these precursors to mature CD56^bright NK cells can be mediated by cytokines and supported by stromal cells (8, 30). However, from these previous observations, it is not yet clear to what extent NK cell differentiation in SLO might account for the total mature NK cell compartment in the body, as most NK cells in human PB are CD56^dim. We now suggest that also the final maturation of CD56^bright into CD56^dim might occur in SLO, further supporting the hypothesis that CD56^dim NK cells might correspond to the terminally differentiated stage of human NK cell development. Although reactive SLO might represent an important site of NK cell differentiation and maturation, such developmental processes could also take place in other inflamed tissues, where both CD56^bright and CD56^dim NK cells can be found (35). Whatever the case, it would be of great interest to investigate which cell type resident in the LN is essential to induce NK cell proliferation and maturation. DCs are interesting candidates because NK cells and DCs are colocalized in LN paracortex and medulla, and have been shown to interact together over extended times (34, 36). Because we have shown previously that both myeloid and plasmacytoid DCs can induce selective expansion of CD56^bright NK cells (36, 37, 38), it would be important to determine whether DCs can induce not only CD56^bright proliferation, but also differentiation into CD56^dim NK cells. To this aim, whether DCs can induce KIR and CD16 expression on proliferating CD56^bright NK cells should be analyzed. It is also not clear at which phase of an immune response NK cell final maturation may happen. Both DC (IL-15 and to some extent IL-12)- and T cell (IL-2)-derived cytokines can induce this differentiation step in vitro. If DC-derived cytokines were primarily involved in NK cell maturation in vivo, this process could take place in the very early phase of an innate immune response before T cell clonal expansion. However, considering the effect of IL-2 in vitro and the significant in vivo association between KIR/CD16 expression and paracortical/follicular hyperplasia, in which extensive lymphocyte proliferation occurs, it is conceivable that NK cell terminal differentiation could take place or be enhanced later on during an immune response, when proliferating naive T cells start to produce high amounts of IL-2.

In conclusion, our data provide new evidence supporting the hypothesis that CD56^bright may give raise to CD56^dim NK cells, and envisage a scenario in which NK cell final maturation and acquisition of competence might occur in SLO during an inflammatory response.

	Acknowledgments

 
We acknowledge Siegfried Kohler for critical reading of the manuscript and discussion; Daniele Marras for inspired critical suggestions; and Toralf Kaiser, Katharina Raba, and Ennio Albanesi for cell sorting.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was funded by grants from Associazione Italiana Ricerca sul Cancro and from Comitato Interministeriale per la Programmazione Economica (02/07/2004, Centro di Biotecnologie Avanzate Project) (to G.Fe.). C.R. is supported by a European Molecular Biology Organization long-term fellowship. B.M. is supported by a fellowship from Fondazione Italiana Ricerca sul Cancro. C.M. is supported by the National Cancer Institute (RO1CA108609).

² Address correspondence and reprint requests to Dr. Guido Ferlazzo, Department of Human Pathology, School of Medicine, University of Messina Via Consolare Valeria, 98125 Messina, Italy; E-mail address: guido.ferlazzo{at}unime.it or Dr. Chiara Romagnani, Clinical Immunology, German Rheumatism Research Centre, Deutsches Rheuma-Forschungszentrum Berlin, Schumannstrasse 21/22, 10117 Berlin, Germany; E-mail address: romagnani{at}drfz.de

³ Abbreviations used in this paper: PB, peripheral blood; DC, dendritic cell; FISH, fluorescence in situ hybridization; KIR, killer Ig-like receptor; LN, lymph node; MFI, mean fluorescence intensity; ORF, open reading frame; PNA, peptide nucleic acid; RTL, relative telomere length; SLO, secondary lymphoid organ.

Received for publication August 10, 2006. Accepted for publication January 19, 2007.

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