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The Journal of Immunology, 2008, 181, 2227 -2237
Copyright © 2008 by The American Association of Immunologists, Inc.
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An Unusual CD56brightCD16low NK Cell Subset Dominates the Early Posttransplant Period following HLA-Matched Hematopoietic Stem Cell Transplantation1

Nicolas Dulphy2,3,*,{dagger},{ddagger}, Philippe Haas2,*, Marc Busson*, Stéphanie Belhadj§, Régis Peffault de Latour, Marie Robin, Maryvonnick Carmagnat*,{ddagger}, Pascale Loiseau*,{dagger}, Ryad Tamouza*,{ddagger}, Catherine Scieux||, Claire Rabian*,{dagger},{ddagger}, James P. Di Santo#,**, Dominique Charron*,{dagger},{ddagger}, Anne Janin{dagger},§, Gérard Socié§,¶ and Antoine Toubert*,{dagger},{ddagger}

* Institut National de la Santé et de la Recherche Médicale, U662, Paris, France; {dagger} Université Paris 7, Institut Universitaire d’Hématologie, Paris, France; {ddagger} Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Louis, Centre d’Investigations Biomédicales "Hématologie Oncologie Greffe", Paris, France; § Institut National de la Santé et de la Recherche Médicale U728, Paris, France; Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Louis, Service d’Hématologie-Greffe de Moelle, Paris, France; || Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Louis, Service de Virologie, Paris, France; # Cytokine and Lymphoid Development Unit, Institut Pasteur, Paris, France; and ** Institut National de la Santé et de la Recherche Médicale U668, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The expansion of the cytokine-producing CD56bright NK cell subset is a main feature of lymphocyte reconstitution after allogeneic hematopoietic stem cell transplantation (HSCT). We investigated phenotypes and functions of CD56bright and CD56dim NK subsets from 43 HLA-matched non-T cell-depleted HSCT donor-recipient pairs. The early expansion of CD56bright NK cells gradually declined in the posttransplant period but still persisted for at least 1 year and was characterized by the emergence of an unusual CD56brightCD16low subset with an intermediate maturation profile. The activating receptors NKG2D and NKp46, but also the inhibitory receptor NKG2A, were overexpressed compared with donor CD56bright populations. Recipient CD56bright NK cells produced higher amounts of IFN-{gamma} than did their respective donors and were competent for degranulation. Intracellular perforin content was increased in CD56bright NK cells as well as in T cells compared with donors. IL-15, the levels of which were increased in the posttranplant period, is a major candidate to mediate these changes. IL-15 serum levels and intracellular T cell perforin were significantly higher in recipients with acute graft-vs-host disease. Altogether, CD56bright NK cells postallogeneic HSCT exhibit peculiar phenotypic and functional properties. Functional interactions between this subset and T cells may be important in shaping the immune response after HSCT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells were originally defined by their capacity to kill tumor cells without prior sensitization (1). We know now that NK cells are crucial components of innate immunity by virtue of their rapid cytokine secretion capacity. Additionally, their role in modulating adaptive immune responses has been increasingly documented (2). In allogeneic hematopoietic stem cell transplantation (HSCT),4 a graft-vs-leukemia (GVL) antitumor effect has been assigned to NK cells of donor origin after transplantation with T cell-depleted transplant from haploidentical donors (3).

NK cells survey infected or abnormal cells through an array of activating or inhibitory receptors that detect the loss of expression of HLA class I molecules or other MHC class I-independent specific cellular signals (4, 5). The combination of these signals triggers and modulates the NK effector functions. Killer cell Ig-like receptors (KIR) are Ig superfamily receptors that can be either activating or inhibitory depending on the presence or absence of ITIM sequences in the intracellular domain. KIR bind specific HLA molecules on target cells, mainly HLA-C and HLA-B (6). CD94/NKG2 heterodimeric isoforms are C-type lectin proteins with either inhibitory (NKG2A) or activating (NKG2C) function. They bind to HLA-E molecules associated with peptides cleaved from the leader sequences of other HLA class I molecules (6, 7). Natural cytotoxicity receptors (NCR: NKp30, NKp44, and NKp46) and NKG2D are capable of activating NK cells to induce the NK-mediated innate immune response (8). NCR belong to the Ig superfamily and are selectively expressed on NK cells. NCR surface expression is variable in different individuals but is also modulated on NK cells from a given donor (8). There is a direct correlation between the ability of NK cells to kill tumor cells and the surface density of expressed NCR (9, 10). Natural ligands for NCR are still undefined. NKp30 and NKp46 are expressed regardless of the activation status of NK cells, while NKp44 is present on activated NK cells (8). Finally, NKG2D, a C-type lectin homodimer, is specific for the stress-inducible MHC class I-related molecules A and B (MICA, MICB) and for UL16-binding protein (ULBP) proteins (4). NKG2D engagement triggers cell-mediated cytotoxicity through the secretion of perforin (11). Interestingly, NKG2D/NKG2D ligand interaction has been implicated in the recognition of leukemia cells by NK cells (12, 13, 14).

According to their phenotype and functional capacities, two NK subpopulations have been defined as CD56brightCD16–/low and CD56dimCD16+ (hereafter referred to as CD56bright and CD56dim NK cells, respectively) (15). In healthy donors, CD56bright NK cells are characterized by a low surface expression of KIR and a high expression of CD94/NKG2A. They express the lymph node-addressing chemokine receptor CCR7. They are enriched in intracellular cytokines such as IFN-{gamma}, TNF-β, or GM-CSF, but they show a low expression of perforin (15). In contrast, CD56dim NK cells have a high expression of KIR, a low expression of CD94/NKG2A, and they express CXCR1, the receptor for the inflammatory cytokine IL-8. A high expression of perforin is observed in CD56dim NK cells for which CD16 (Fc{gamma}RIII) at the cell surface is important to mediate the Ab-dependent cellular cytotoxicity (ADCC) (15). Recent findings suggest that CD56bright NK cells are immature precursors of the CD56dim NK population (16, 17, 18).

The large heterogeneity of the NK subpopulations repertoire may be of potential interest for allogeneic HSCT in the context of developing novel adoptive immunotherapy strategies. Data concerning the NK reconstitution in allogeneic HSCT is still relatively scarce (19, 20, 21). Considering the dichotomy between CD56bright and CD56dim NK cell subsets, our aim was to assess the characteristics of reconstitution and functional properties of CD56bright and CD56dim NK cells in non-T cell-depleted allogeneic HSCT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients and samples

Forty-three donor/recipient pairs (donor mean age of 32 years, range of 7–60 years; recipient mean age of 32 years, range of 6–54 years) transplanted at the Bone Marrow Transplantation Unit, Saint-Louis Hospital (Paris, France) for hematological malignancy (44.2% for lymphoid-derived diseases and 55.8% for myeloid-derived diseases; see Table I for details) between January 2000 and August 2004 were included in this study. Patients received only unmanipulated bone marrow or peripheral blood stem cells. Table I summarizes the characteristics of the population studied.


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  		Table I. Patient and donor characteristics

 
Blood samples were collected from donors and from recipients 15 days before transplant and at 3, 6, and 12 mo after transplant. PBMCs were isolated from freshly collected blood samples by density gradient centrifugation using lymphocyte separation medium (Eurobio) and stored in liquid nitrogen.

Tissue samples originated from upper digestive tract biopsies performed for diagnostic purposes. During the endoscopic procedure, two biopsy samples were immediately fixed in formaldehyde for further paraffin embedding, and two biopsy samples were immediately snap frozen. When the diagnosis had been established, the remaining tissue was used for this study. Controls were histologically normal digestive samples harvested in macroscopically normal areas in surgical pieces of colon carcinoma.

All patients gave their informed consent to this study, which was approved by the Institutional Review Board of the Hospital Saint-Louis (Paris, France).

Abs and flow cytometry analysis

Lymphocyte immunophenotyping was performed on frozen samples of PBMCs in four-color analysis on a BD LSR flow cytometer (BD Biosciences). The following Abs were used: CCR7-FITC, CD122-FITC, and NKG2C-PE produced by R&D Systems; CD16-FITC, NKp30-PE, NKp44-PE, NKp46-PE, NKG2A-PE, NKG2D-PE, and CD117-allophycocyanin from Beckman Coulter; CD57-FITC, CD94-FITC, CD158a (HP-3E4)-FITC, CD158b (CH-L)-FITC, CD158e (NKB1)-FITC, CD161-FITC, CXCR1-FITC, CD16-PE, TRAIL-PE, CD3-PerCP, CD56-allophycocyanin, CD127-allophycocyanin, CD3-allophycocyanin/Cy7, and CD56-biotin/streptavidin-PerCP produced by BD Biosciences. The IL-2R{alpha} expression was determined with CD25-PE (Miltenyi Biotec). Intracellular perforin was detected with the perforin-FITC set (BD Biosciences). IFN-{gamma} detection was performed by intracellular staining with a FITC-labeled Ab (BD Biosciences) on NK cells fixed with a 2% paraformaldehyde-PBS solution and permeabilized with saponin (0.1%). All flow cytometry analyses were performed with a combination of CD3, CD16, and CD56 Abs associated to a fourth specificity to gate specifically either CD3+ T lymphocytes or the CD3–CD56brightCD16low/– and CD3–CD56dimCD16bright NK populations. T lymphocytes were studied with the following combinations: CD62L-FITC/CD45RA or CD45RO-PE/CD4-PerCP/CD8-allophycocyanin (BD Biosciences), and CD62L (Beckman Coulter). Absolute counts of total, CD4+, and CD8+ lymphocytes were determined with the BD MultiTest CD3-FITC, CD8-PE, CD45-PerCP, CD4-allophycocyanin (BD Biosciences).

KIR and HLA genotyping

Eleven KIR (KIR 2DL1, 2DL2, 2DL3, 3DL1, 3DL2, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, and 3DS1) were typed according to the technique and with the primers previously described by Uhrberg et al. (22). HLA class I (A, B, and C) and II (DRB1, DQB1, and DPB1) typing was performed with the PCR sequence-specific oligonucleotide reverse dot-plot kits from Innogenetics (Inno-Lipa kits). Definition of HLA-C1 and HLA-C2 groups was attained by molecular HLA-C typing as previously described (23).

NK cell enrichment

NK cells were enriched by negative selection using the NK cell isolation kit II from Miltenyi Biotec. NK cells were cultured overnight in culture medium RPMI 1640 supplemented with 10% FCS without cytokines before use in degranulation assays.

CD107a degranulation, IFN-{gamma} production, and redirection assays

Target cells K562, C1R, or C1R transfected with the NKG2D ligand MICA (24) were incubated with sorted NK cells at an E:T ratio of 1:1 for 6 h at 37°C in culture medium supplemented with CD107a-FITC (BD Biosciences) and BD GolgiStop according to the manufacturer’s recommendations (BD Biosciences). Thereafter, cells were incubated with CD16-PE, CD3-PerCP, and CD56-allophycocyanin Abs for 20 min on ice. Cells were fixed in PBS-1% paraformaldehyde and analyzed by flow cytometry. The CD3-specific staining ensured elimination of any signal from potential contaminating T lymphocytes.

IFN-{gamma} produced by NK cells after stimulation either with target cells or IL-12 (10 ng/ml) and IL-18 (100 ng/ml) as previously described (21) was quantified by intracellular staining after 24 h of incubation at 37°C with 5 ng/µl of brefeldin A (Sigma-Aldrich) for the last 5 h.

Redirection assays (25, 26) were set up with P815 as target cells preincubated with mAbs (5 µg/ml) specific for NKG2D, NKp46, and NKG2A (R&D Systems) or with IgG1 (Sigma-Aldrich) and IgG2a and IgG2b controls (R&D Systems). Then, P815 cells were washed twice and incubated with purified NK cells at an E:T ratio of 1:2. Cell-surface CD107a or intracellular IFN-{gamma} were detected as described above.

Immunohistochemistry analysis

Digestive biopsies of 12 patients (all full hematopoietic donor chimeras) without relapse at the time were studied. The biopsies had been taken before any high-dose steroid therapy, and all patients showed histological signs of graft-vs-host disease (GVHD) in the samples studied.

An indirect immunoperoxidase method on an automated device (NexEX from Ventana Medical Systems) was used on 5-µm-thick frozen sections. Abs directed against CD3 (DakoCytomation), perforin (BioVision), and NKG2D and MICA (R&D Systems) were used as primary Abs. The specificity of immunohistochemistry was tested with irrelevant isotype-matched Abs as negative controls. Tissue sections were blindly analyzed on an Olympus Provis AX70 microscope. Antibody-specific positive cell counting was performed in three different fields at magnification x400 (optic type: 40, UPlan F1, numerical overture: x40/0.75). At this magnification, the field size was 0.344 mm2.

IL-15 ELISA assay

IL-15 was quantified by ELISA in recipients’ serum at transplantation days –15, 50, and 90 and in donors with the Quantikine kit (R&D Systems) according to the manufacturer’s instructions.

Statistical analysis

Nonparametric Wilcoxon tests were used to compare continuous variables between two groups. To test differences between donors and recipients or among recipients, a Wilcoxon rank sum test was used. A Wilcoxon signed-rank test compared cell populations from the same samples. All correlations were assessed with a Spearman rank correlation test. Univariate Kaplan-Meier analysis was used in combination with the log-rank test to compare the incidence of acute GVHD (aGVHD) between two groups of recipients. There were no censored data in these series. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD56bright NK cell expansion after allogeneic HSCT

Recipient NK cell phenotype and function were analyzed and compared with the donor NK cells for a 1-year period following HSCT. The frequency of NK cells in lymphocytes (but not absolute number) was increased in HSCT recipients during the first 3 mo after transplant compared with HSCT donors. This increase was still present 6 mo after transplantation. However, we observed that the equilibrium between the two CD56bright and CD56dim NK subsets was disturbed for at least 1 year after transplantation (Fig. 1, A–D and Table II).


Figure 1
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  		FIGURE 1. Sustained CD56bright NK cell expansion after transplant. A–C, The percentages of total (A), CD56bright (B), and CD56dim (C) NK cells among lymphocytes were measured by flow cytometry in donors and in recipients 3, 6, and 12 mo after transplant. D, Percentage of CD56bright NK cells among total NK cells. E, Representative stainings of PBMCs from one donor/recipient pair. Plots were gated on a CD3– lymphocyte gate, and the numbers indicate the percentages of CD56bright and CD56dim NK cells among the total NK population. Horizontal bars indicate the medians. ***, p < 0.001 for comparisons with donors.

 

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  		Table II. NK cell phenotype in donors and recipients after transplantationa

 
Frequency of the CD56bright population in the periphery was increased 3 mo after transplant (median: 5.65% of recipients’ PBL vs 0.54% of donors’ PBL, p < 10–9, Fig. 1B), whereas percentages of CD56dim NK cells were comparable to donors (6.09% of recipients’ PBL and 4.15% of donors’ PBL, Fig. 1C). There was no correlation between NK subsets in donors and recipients 3 mo posttransplant (data not shown), and there was no impact of donor or recipient CMV serology on the balance between CD56bright and CD56dim populations (data not shown). Concerning the absolute number of each subset, the difference was even more striking, as CD56bright cell counts were increased (median: 43/mm3 in recipients and 14/mm3 in donors), whereas the CD56dim population was reduced (37/mm3 in recipients and 104/mm3 in donors). The median percentage of CD56bright cells among NK cells in the periphery, which was 12.3% in donors (from 2.47% to 33.6%), reached 54.89% (from 3.55% to 93.82%, p < 10–9) in recipients at 3 mo after transplant. Then, it gradually decreased but was still increased at 1 year after transplant (median: 28.73%, from 7.84% to 91.1%, p < 10–5, Fig. 1, D and E). Comparisons of frequencies and absolute counts of NK subsets showed that NK early recovery after allogeneic HSCT was due to a considerable expansion of the CD56bright population that was maintained for the year following the transplant.

Both CD56bright and CD56dim subsets may be characterized by a panel of surface markers (15, 27). We observed some variations in frequencies and levels of expression of several markers 3 mo after transplantation compared with HSCT donors. These changes still persisted 1 year after transplant (Fig. 2 and Table II).


Figure 2
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  		FIGURE 2. Phenotypic alterations of CD56bright and CD56dim NK cell subsets after transplant. The percentage of expression of NK markers was measured independently on both NK cell subsets of peripheral blood from donors and recipients at 3, 6, and 12 mo after transplant. Both panels depict the expression of NK markers on donor and recipient CD56bright (A) and CD56dim (B) NK cells. Each horizontal line is dedicated to a definite NK marker, with the color of each square reflecting the percentage of expression of the corresponding marker in a NK cell subset of a given blood sample. The values measured for donor and recipient samples were color displayed and rank ordered considering the donors’ median as a reference: blue indicates inferior to median, yellow indicates superior, with the use of the Genesis program (available at www.genome.tugraz.at) (55 ). Missing values are displayed in gray. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for comparisons with donors.

 
KIR expression and genotype/phenotype comparisons

The presence or absence of KIR gene-related proteins in allogeneic HCST recipients after transplant was always identical to what was observed in the corresponding donor, showing that recipient KIR expression was directly related to donor KIR genotype (Fig. 3Ai). More specifically, there was a positive correlation between donor and recipient KIR expression frequency on both CD56-expressing NK subsets.


Figure 3
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  		FIGURE 3. KIR, lectin-like receptors, and perforin expression after transplant. A, HLA genotyping and KIR genotyping and phenotyping were performed on donors and recipients. i, Flow cytometric expression of KIR3DL1 on total NK cells of two donor/recipient pairs. The areas above the dot plots indicate donor and recipient KIR3DL1 genotypes. ii, CD158b expression in CD56dim NK cells was measured by flow cytometry and displayed as a function of the presence of its ligand HLA-C1. Top panel, Donors. Bottom panel, Recipients 3 mo after transplantation. iii, Variations of KIR expression in total NK cells parallel the CD56dim/CD56bright equilibrium. Top panel, Open circles, filled diamonds, and black bars indicate the median percentages of CD158b expression on CD56bright, CD56dim, and total NK cells, respectively. Bottom panel, Median percentages of CD56dim and CD56bright subsets among total NK cells. B, Sustained overexpression of lectin-like receptors and perforin after transplant. The histograms shown are gated on a CD56bright NK cell gate and are representative of 27 patients. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
There is compelling evidence that HLA genotype conditions KIR expression (28). Accordingly, CD158b expression on donor NK subpopulations was correlated to the number of CD158b ligand alleles (HLA-C S-77/N-80 molecules (4)) in the donor HLA genotype (p = 0.003 for CD56bright and p = 0.049 for CD56dim NK cells). Interestingly, this correlation was not observed in recipients after transplant despite the HLA matching between donors and recipients (Fig. 3Aii). This observation emphasizes the particular environment surrounding HSCT and its impact on NK repertoire reconstitution independently of KIR and HLA genotypes.

KIRs were present on recipient CD56dim NK cells at the same level as in donors, while CD56bright cells overexpressed CD158e (5.25% in recipients and 3.18% in donors, p = 0.018) and only moderately underexpressed CD158b (4.49% in recipients and 7.0% in donors, p = 0.08). However, the result on the whole NK population was a very significant decrease of CD158b expression (17.62% in recipients and 31.36% in donors, p < 10–5, Fig. 3Aiii), while CD158a and CD158e were not significantly modified. The reduced frequency of CD158b+ cells after allogeneic HSCT is indeed explained by the expansion of the low KIR expressing CD56bright NK subset in the recipients’ periphery.

Activating and lectin-like NK receptors

Recipient CD56bright cells overexpressed the inhibitory heterodimer CD94/NKG2A and the activating receptors NKG2D and NKp46 but not NKp30 and NKp44 as compared with donor CD56bright cells (Figs. 2A and 3B). NKG2C expression was not perturbed on either of the two NK subsets. Most of the studied markers present on CD56dim NK cells showed a similar level of expression after transplantation as compared with donors except for NKp46 and CD94/NKG2A, which were increased in recipients. Intracellular perforin was expressed in the vast majority of NK cells, and the frequency of perforin-positive cells together with its expression level were significantly increased in the CD56bright population compared with donors (Fig. 2A). NKp46, NKG2D, and NKG2A expressions appeared to be correlated together on CD56bright cells, suggesting that the regulation of these three markers may share common stimuli (data not shown). Conversely, KIR expression was inversely correlated with NKG2A, NKp46, and NKG2D expression on the CD56bright subset (data not shown).

CD56bright NK cells show an increased production of IFN-{gamma} after allogeneic HSCT

First clues of the NK activity are the content in intracellular perforin and the capacity to produce cytokines. IFN-{gamma} production was evaluated in both CD56bright and CD56dim subsets (Fig. 4A) after a nonspecific stimulation by a combination of IL-12 and IL-18. The ability of the 3-mo posttransplant recipient NK cells to produce IFN-{gamma} was increased as compared with their respective donors (35.11% vs 20.50%, p = 0.006). This observation was the result of the enhanced IFN-{gamma} production by recipient CD56bright cells (87.44% vs 74.50%, p = 0.027) combined with the expansion of the CD56bright subset, whereas IFN-{gamma}+CD56dim cells were comparable between recipients and donors. Altogether, increased intracellular perforin and IFN-{gamma} production favored a strong capacity of recipient CD56bright populations to provide support to the adaptive immune response.


Figure 4
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  		FIGURE 4. NK cell function after transplant. A, Donor (n = 17) or 3-mo postgraft recipient (n = 17) PBMCs were incubated for 24 h in IL-12 (10 ng/ml) and IL-18 (100 ng/ml). The percentage of IFN-{gamma}+ CD56bright and CD56dim NK cells was subsequently determined by flow cytometry. B, Donor (n = 10) or 3-mo posttransplant recipient (n = 13) NK cells were negatively sorted and then incubated for 6 h either alone or with K562 cells at an E:T ratio of 1:1. Degranulation was assessed as the percentage of CD107a+ CD56bright or CD56dim NK cells. Plots show an experiment with a recipient and were gated on a NK cell gate. C, Healthy subject (n = 6) or 3-mo postgraft recipient (n = 6) NK cells were negatively sorted and incubated at an E:T ratio of 1:1 with P815 cells in redirected assays using isotype control, anti-CD16, or anti-NKG2A mAbs. Plots show an experiment with a recipient and were gated on a NK cell gate. D, As in C, healthy subject (n = 6) or 3-mo posttransplant recipient (n = 6) NK cells were negatively sorted and incubated at an E:T ratio of 1:1 with P815 cells in redirected assays using the specified mAbs. i, Plots show an experiment with a recipient and were gated on a NK cell gate. ii, Percentage of recipient CD107a+ CD56bright and CD56dim NK cells (= %CD107a+ cells with mAbs of interest minus %CD107a+ cells with isotype control mAbs). iii, Percentage of recipient IFN-{gamma}+ CD56bright and CD56dim NK cells (= %IFN-{gamma}+ cells with mAbs of interest minus %IFN-{gamma}+ cells with isotype control mAbs). Negative values are displayed as equal to 0. Black bars indicate the medians. *, p < 0.05 for comparisons with donors.

 
Receptors overexpressed in CD56bright NK cells after transplant interact to modulate degranulation of cytotoxic granules

We asked whether the receptors whose expression was increased after transplant in CD56bright cells might have an impact on NK activation. Three months after transplant, sorted recipient NK cells were fully able to kill the NK target K562 in a classical 51Cr-release assay (data not shown), but this method was not suitable to evaluate each subset individually. Because it was not feasible to sort the NK subsets in transplant samples, we used a flow cytometry-based assay to discriminate between the degranulation of each NK subset by detecting cell surface expression of the lysosomal-associated membrane protein CD107a (LAMP-1) (25, 26). To ensure that we analyzed the functions of resting cells, NK lymphocytes were negatively sorted and did not receive any cytokine-mediated stimulation before the assay. NK cell degranulation activity in donors or recipients 3 mo after transplant was evaluated upon stimulation by K562 (Fig. 4B). CD56bright NK cells showed a stronger CD107a mobilization than did CD56dim cells either in donors or recipients. More importantly, CD56bright and CD56dim cells from recipients were as efficient as donor cells to mobilize CD107a onto their surface (data not shown), demonstrating that both recipient NK populations are fully functional. This was also confirmed by activating the CD16 pathway (Fig. 4C).

Phenotype analysis showed an increased expression of the activating proteins NKp46 and NKG2D together with perforin on the one hand and of the inhibitory receptor NKG2A on the other hand in CD56bright cells. This raised the issue of the respective roles of these receptors in the degranulation capacity of CD56bright NK cells.

We thus investigated the role of the interactions between NKG2D, NKp46, and NKG2A in regulating NK functions after transplant in redirected CD107a mobilization assays against the P815 murine mastocytoma cell line (Fig. 4, Di and Dii). NKG2D alone was capable of providing a weak activation signal to NK cells, whereas NKp46 moderately stimulated degranulation by CD56bright as well as by CD56dim populations. These two activating receptors strongly synergized NK cell degranulation but more efficiently in CD56bright than in CD56dim cells (p = 0.031). IFN-{gamma} production was also evaluated in redirection assays (Fig. 4Diii). IFN-{gamma} was inducible in CD56bright but not in CD56dim cells, consistent with the role of cytokine producer attributed to the CD56bright subset (15). NKp46 alone was weakly capable of inducing IFN-{gamma} production, whereas NKG2D stimulation alone was inefficient, but, here again, both receptors synergized for IFN-{gamma} production. Importantly, the simultaneous stimulation of NKG2A almost completely abrogated the signals provided by the NKp46 and NKG2D receptors for degranulation as well as IFN-{gamma} production. These results highlight the interactions between the activating receptors NKG2D and NKp46 and the inhibitory receptor CD94/NKG2A in posttransplant CD56bright cells.

Identification of an intermediate CD56brightCD16low NK population undergoing maturation

Repeatedly, we detected among the CD56bright population a CD56brightCD16low subset in addition to the original CD56brightCD16– subset (Fig. 1E). This population was significantly increased in recipients compared with donors (25.32% of recipient NK cells at 3 mo vs 5.06% of donor NK cells, p < 10–10) and constituted up to 61% of the CD56bright population at 6 mo after transplant (Fig. 5A). In fact, the decrease of the CD56bright population in recipients observed for the year after transplant was, in first, due to the CD56brightCD16– subset, whereas the CD56dimCD16+ population was still increasing. These observations would suggest a process where the CD56brightCD16– cells are undergoing differentiation toward the CD56brightCD16low subset, an intermediate population before the terminally differentiated CD56dimCD16+ NK cells.


Figure 5
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  		FIGURE 5. Characterization of a posttransplant intermediate CD56brightCD16low NK cell population. A, Median percentages of CD56brightCD16–, CD56brightCD16low, and CD56dimCD16+ subpopulations among total NK cells. ***, p < 0.001 for comparison with donors. B, Expression of markers in CD56brightCD16–, CD56brightCD16low, and CD56dimCD16+ NK cells 3 mo after transplant: NKG2D (n = 37); CD158e (n = 30); perforin (n = 36); CD25, CD117, and TRAIL (n = 12). The medians are shown, and the error bars stand for the interquartile range. MFI indicates median fluorescence intensity. **, p < 0.01; ***, p < 0.001.

 
Indeed, the CD56brightCD16low cells showed NKG2D, KIR, and perforin expression levels that were intermediate between the CD56brightCD16– and the CD56dimCD16+ NK cells, although closer to the CD56brightCD16– subset (Fig. 5B). Additional markers, which decrease along normal NK ontogeny, were analyzed (29). These include cytokine receptors involved in the maturation and proliferation of the NK progenitors (CD25, CD117, CD122, and CD127) and TRAIL, a surface marker of NK cytotoxic activity. We observed in recipients some differences in agreement with an ongoing maturation from the CD56brightCD16– cells to the CD56dimCD16+ NK cells (Fig. 5B): CD122 and CD127 were expressed at the same level as in the CD56brightCD16– population (data not shown), whereas CD25, CD117, and TRAIL were expressed at an intermediate level.

T and NK lymphocyte reconstitution after allogeneic HSCT suggests an IL-15-driven homeostatic process

NK phenotype and function suggest an ongoing cytokine-driven process of maturation in allogeneic HSCT recipients. IL-15 is the paramount cytokine in NK differentiation and in NK and T cell homeostasis (30), inducing a high proliferation rate of CD56bright NK cells. Additionally, some data show the implication of IL-15 not only in NK proliferation (31, 32, 33) but also in the occurrence of GVHD (34, 35). IL-15 may also increase NKG2D cell surface expression (36) and the synthesis of effector molecules such as perforin in CD8+ memory T cells (37). This suggested that some features of NK and T cell reconstitution in HSCT patients could be IL-15 driven. Because NK receptors may be expressed on T lymphocytes and modulate their function, we assessed the different NK markers in CD3+ T cells as well as in NK populations. By comparing donors and recipients 3 mo after transplant (Fig. 6), we found an increase in NKG2A+ (2.26% and 6.56% respectively, p < 10–3), NKG2D+ (32.27% and 44.47%, respectively, p < 10–4), and perforin+ T cells (17.51% and 44.03%, respectively, p < 10–3). NKG2D expression level was positively correlated between T cells and CD56bright NK cells (p < 10–6). The same was observed for perforin expression (p < 10–3).


Figure 6
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  		FIGURE 6. Enhanced expression of NK cell markers by T cells after transplant. A, Representative stainings of PBMCs from one donor/recipient pair. Plots were gated on a CD3+ lymphocyte gate. B, NKG2A, NKG2D, and intracellular perforin expression in T cells were measured by flow cytometry in donors and recipients 3 mo after transplant. Horizontal bars indicate the medians. ***, p < 0.001 for comparisons with donors.

 
IL-15 concentration in the serum of recipients was quantified before transplantation and 50 and 90 days after transplantation (Fig. 7). The concentration of IL-15 increased after transplant and correlated positively with the frequency of CD8+ T cells (p = 0.014). The IL-15 burst observed after transplant (Fig. 7A), and perforin levels in T cells were associated with the clinical outcome: IL-15 levels were higher in case of aGVHD grades 2–4 compared with grades 0–1 (p = 0.033, Fig. 7B) and IL-15 of <2 pg/ml before transplant was predictive of a lower aGVHD incidence (p = 0.004, Fig. 7C). Finally, higher perforin contents in CD3+ T cells correlated with grades 2–4 vs grades 0–1 aGVHD (p = 0.035).


Figure 7
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  		FIGURE 7. Rise of serum IL-15 after transplantation and association of pretransplant serum IL-15 with aGVHD. The concentration of IL-15 was measured by ELISA in the serum of donors (n = 31) and recipients before transplant (n = 29) and 50 days (n = 6) or 90 days (n = 34) after transplant. A, IL-15 serum concentrations were raised after transplant. B, Acute GVHD was associated with higher IL-15 serum concentrations at day 90 posttransplant. C, Kaplan-Meier analysis of aGVHD incidence. Patients were categorized in two groups according to pretransplant IL-15 level: "low" IL-15 (IL-15 of <2 pg/ml, n = 15) and "high" IL-15 (IL-15 of >2 pg/ml, n = 14). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
Increased expression of MICA on epithelial cells of aGVHD intestinal lesions and presence of NKG2D+ cells in the T cell infiltrate

We showed that recipient NK and T cells express high levels of NKG2D in the periphery. We therefore asked how NKG2D and its ligand MICA were expressed in intestinal biopsies of aGVHD patients. Intestinal biopsies from aGVHD patients (n = 12) were studied by immunohistochemistry and showed a strong expression of MICA on epithelial cells compared with normal intestinal tissue (Fig. 8, A and B). Histochemistry analysis showed an infiltrate of CD3+ T cells, NKG2D expressing cells, and perforin+ cells at intralesional sites (Fig. 8, C–E). Quantification of the infiltrate showed a correlation between the presence of CD3+ lymphocytes and NKG2D+ cells (p = 0.001). These data were consistent with the above-mentioned phenotypic observations in the periphery and with the correlation of perforin-expressing T cells and aGVHD.


Figure 8
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  		FIGURE 8. Immunohistochemical analysis of intestinal aGVHD lesions. MICA expression on epithelial cells in graft-vs-host biopsy (A) compared with normal colonic epithelium used as control (B). The aGVHD cellular infiltrate contains cells expressing CD3 (arrows in C), NKG2D (arrows in D), and perforin (arrows in E). E, inset, Granular cytoplasm of immunostained cell at higher magnification.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite current data showing a strong GVL effect mediated by donor-origin NK cells after transplant (3, 38), the degree of maturity and the functionality of NK cells after allogeneic HSCT remain controversial. Apparently conflicting results could be due to differences in the transplant setting, including the source of stem cells, T cell depletion or not, haploidentical vs HLA genoidentical-related donors or HLA-matched unrelated donors, as well as the experimental approach implemented (19, 20, 21). This study, conducted prospectively in a series of non-T cell-depleted HLA-matched transplants, could clarify some points by taking advantage of a comprehensive analysis, both phenotypically and functionally, of the two major NK subsets separately, namely CD56bright and CD56dim NK cells.

The NK repertoire observed in recipients was intimately associated with the imbalance between CD56bright and CD56dim NK subsets, which drives perturbations in the expression of markers such as KIR or lectin receptors in the whole population. Compared with their donors, CD56bright cells displayed in the recipients a high expression of three regulatory receptors, NKp46, NKG2D, and CD94/NKG2A, and of the intracellular perforin. By contrast, the CD56dim population was similar to donors, with the exception of an increased NKG2A and NKp46 expression. Taken together, this resulted in an increased expression of CD94/NKG2A and a reduced expression of KIR on the whole NK population. Functionally, despite these changes, CD56bright NK cells produced large amounts of IFN-{gamma}, and both subsets taken separately had a degranulation propensity comparable to normal NK cells, including the synergy of NKG2D- and NKp46-activating signals (26). Therefore there was no evidence, at least in this setting and in contrast to haploidentical T cell depleted transplants (19), for globally impaired NK functions. Functional behavior of CD56bright and CD56dim NK subsets may rather be regulated by the expression level of the respective NK receptor ligands on target cells, such as MICA.

The CD56bright expansion should be considered a key feature in NK reconstitution. In healthy adults, NK cells differentiate in bone marrow from a common lymphoid precursor (31). However, recent studies have suggested that different sites could produce CD56bright NK cells. Freud et al. described CD34dimCD45RA+ hematopoietic stem cells with high levels of the integrin {alpha}4β7 as CD56bright NK cell precursors in human adult peripheral blood and lymph nodes (39). The CD34dim precursor differentiate to a CD56bright-like subset through intermediates characterized by the expression of CD117lowCD94+ and GATA-3+ (40). Lymph node CD56bright NK cells are located in the paracortical T cell-rich regions (2, 41) where CD34dim precursors could interact with surrounding T cells, and probably dendritic cells, to differentiate. Additionally, Vosshenrich et al. (42, 43) recently suggested that the thymus could be a site of CD56bright NK cell differentiation in humans. Thymus-derived NK populations expressed high levels of CD127 and GATA-3. Altogether, these results suggest specific but nonunivocal differentiation pathways for CD56bright cells, which warrant further studies in the context of allogeneic HSCT. Thereafter, we could observe ex vivo a pattern consistent with an ongoing NK maturation from CD56bright precursors to a CD56dim mature population, as shown by the expansion of the CD56brightCD16low subset. This progression was assessed by the apparent progressive transfer of cells from the CD56brightCD16– compartment to the CD56brightCD16dim and finally to the CD56dimCD16+ subset, but also by the reduction of the expression of markers such as CD25, CD117, or TRAIL, as described by Huntington et al. (29). Indeed, the proposed progression of CD56bright to CD56dim NK cell development is supported by in vivo and in vitro observations of human NK cell differentiation (16, 17, 18).

The phenotypic and functional pattern of CD56bright NK cells after transplant is suggestive of a cytokine-driven process. Some of the cytokines (e.g., IL-2 and especially IL-15) produced during the conditioning regimen and early after transplant are key factors in the differentiation and peripheral homeostasis of NK cells together with molecules such as IL-12 and IL-18 (31, 32, 33). IL-15 is produced by activated dendritic cells, monocytes, and stromal cells. CD56bright cells are very sensitive to IL-2 and IL-15 because they constitutively express the high-affinity heterotrimeric IL-2 receptor, which shares two chains (IL-2Rβ and {gamma}c) with the IL-15 receptor (27, 44). In vivo, soluble IL-15 is bound on IL-15R{alpha} on IL-15R{alpha}+ accessory cells and presented in trans to IL-15Rβ+{gamma}c+ cells (45, 46). Trans-presentation of IL-15 is essential for mature NK cells to differentiate and survive (27, 44). IL-15 is also known to induce NKG2D surface expression (36) and to enhance the production of IFN-{gamma} by NK cells (47). Synergy between IL-15, IL-12, and IL-18 appears to strongly activate CD56bright NK cells (48). These physiological properties of IL-15 may explain our observations in vivo in the context of allogeneic HSCT lymphopenia. The lack of a direct correlation between IL-15 levels in the sera and NK cell counts could be due to the paracrine in trans mode of action of IL-15/IL-15R{alpha} complexes. This could account for the persistent in vivo effect of IL-15 that also helps survival of CD8 T cells (49), a correlation we observed herein. IL-15 levels markedly increased after transplant. In this cohort of patients, high IL-15 levels were associated with aGVHD, but we were unable to evaluate the clinical impact on GVL due to a low relapse rate. Interestingly, in murine models, IL-15 could also have an impact on immune reconstitution with a dual effect on GVL and aGVHD (34, 35, 50). Especially, IL-15 administration could increase CD8+ T cell and NK cell proliferation and exacerbate aGVHD in recipients of T cell-depleted bone marrow transplants (50). Th1 priming was necessary for IL-15-mediated aGVHD lethality (35).

In healthy individuals, CD56bright cells are preferentially found in lymph nodes, where they interact with dendritic cells and T cells. IL-15 trans-presentation by dendritic cells in lymph nodes is crucial for NK priming (51). NK cells in lymph nodes enhance Th1 activation via IFN-{gamma} secretion in mice (52) as well as in humans (53). In turn, T cell-derived IL-2 stimulates CD56bright NK cells to produce IFN-{gamma} through the high-affinity IL-2 receptor. We could clearly observe an increase of the posttransplant CD56bright cell capacity to produce IFN-{gamma} upon IL-12/IL-18 stimulations as compared with donors. Therefore, the hypothesis we would favor is that CD56bright NK cells get activated by IL-15, probably through trans-presentation in vivo by dendritic cells and/or monocytes. In turn, within lymph nodes, activated NK cells would trigger alloreactive T cells with a GVL and/or aGVHD potential. The up-regulation of the NKG2D ligand MICA at the surface of intestinal epithelial cells would provide a costimulatory signal to such alloreactive CD8 T cells in the GVHD direction. On the other hand, the endogenous IL-15 burst within the early period after transplant could be helpful to optimize adoptive antitumoral NK immunotherapy as reported in a nontransplantation setting (54).


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts 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 supported by research grants from the Association de Recherche contre le Cancer (N.D.), Fondation pour la Recherche Médicale (fellowship DEA20040902110 to P.H.), Association Laurette Fugain, Cancéropôle Ile-de-France, and EC program FP6 ALLOSTEM (no. 503319). Back

2 N.D. and P.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Nicolas Dulphy, Unité Institut National de la Santé et de la Recherche Médicale U662, Centre Hayem, Institut Universitaire d’Hématologie, Hôpital Saint-Louis, 1, avenue Claude Vellefaux, F-75010, Paris, France. E-mail address: nicolas.dulphy{at}univ-paris-diderot.fr Back

4 Abbreviations used in this paper: HSCT, hematopoietic stem cell transplantation; ADCC, antibody-dependent cellular cytotoxicity; aGVHD, acute GVHD; GVHD, graft-vs-host disease; GVL, graft vs leukemia; KIR, killer immunoglobulin-like receptor; MICA/B, MHC class I-related molecules A and B; NCR, natural cytotoxicity receptor; ULBP, UL16-binding protein. Back

Received for publication November 8, 2007. Accepted for publication May 24, 2008.


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 Introduction
 Materials and Methods
 Results
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