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Expansion of CMV-Mediated NKG2C+ NK Cells Associates with the Development of Specific De Novo Malignancies in Liver-Transplanted Patients
Abla Achour, Florence Baychelier, Caroline Besson, Armelle Arnoux, Michel Marty, Laurent Hannoun, Didier Samuel, Patrice Debré, Vincent Vieillard and the K-GREF Study Group
J Immunol 2014; 192:503-511; Prepublished online 4 December 2013; doi: 10.4049/jimmunol.1301951 http://www.jimmunol.org/content/192/1/503

Supplementary Material
References

http://www.jimmunol.org/content/suppl/2013/12/05/jimmunol.130195 1.DC1
This article cites 48 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/192/1/503.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Expansion of CMV-Mediated NKG2C+ NK Cells Associates with the Development of Specific De Novo Malignancies in Liver-Transplanted Patients

Abla Achour,*,†,1 Florence Baychelier,*,†,1 Caroline Besson,‡ Armelle Arnoux,x Michel Marty,{ Laurent Hannoun,‖ Didier Samuel,#,** Patrice Debre´,*,† Vincent Vieillard,*,†
and the K-GREF Study Group

Solid cancers are a major adverse outcome of orthotopic liver transplantation (OLT). Although the use of chronic immunosuppression is known to play a role in T cell impairment, recent insights into the specificities of NK cells led us to reassess the potential modulation of this innate immune cell compartment after transplantation. Our extensive phenotypic and functional study reveals that the development of specific de novo noncutaneous tumors post-OLT is linked to unusual NK cell subsets with maturation defects and to uncommon cytokine production associated with the development of specific cancers. Remarkably, in CMV+ patients, the development de novo head/neck or colorectal tumors is linked to an aberrant expansion of NK cells expressing NKG2C and a high level of intracellular TNF-a, which impact on their polyfunctional capacities. In contrast, NK cells from patients diagnosed with genitourinary tumors possessed a standard immature signature, including high expression of NKG2A and a robust production of IFN-g. Taken together, our results suggest that under an immunosuppressive environment, the interplay between the modulation of NK repertoire and CMV status may greatly hamper the spectrum of immune surveillance and thus favor outgrowth and the development of specific de novo tumors after OLT. The Journal of Immunology, 2014, 192: 503–511.

O rthotopic liver transplantation (OLT) provides life-saving therapy for patients with end-stage liver diseases. The categories of fatal liver diseases that may be treated by liver grafting include primary cancers such as hepatocarcinoma (HCC) and severe nonmalignant destructive liver cell diseases, including cirrhosis, fulminant hepatic failure, and chronic hepatitis infection, which remains a leading cause of liver-related mortality despite the development of effective antiviral agents in the past decade (1, 2). Although transplant outcomes have improved, de
*INSERM, Unite´ Mixte de Recherche S945, Laboratoire Immunite´ et Infections, F-75013 Paris, France; †Universite´ Pierre et Marie Curie Paris 06, Unite´ Mixte de Recherche S945, F-75005 Paris, France; ‡Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Biceˆtre, Service d’He´matologie, F-94270 Le Kremlin Biceˆtre, France; xAssistance Publique-Hoˆpitaux de Paris, Hoˆpital Biceˆtre, Unite´ de Recherche Clinique Paris Sud, F-94270 Le Kremlin Biceˆtre, France; {Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Saint-Louis, Centre des Innovations The´rapeutiques en Oncologie et He´matologie, F-75010 Paris, France; ‖Assistance Publique-Hoˆpitaux de Paris, Hoˆpital La Pitie´-Salpeˆtrie`re, Service de Chirurgie Digestive et He´pato-bilio-pancre´atique, F-75013 Paris, France; #Assistance Publique-Hoˆpitaux de Paris, Centre He´patobiliaire, Hoˆpital Paul-Brousse, INSERM Unite´ Mixte de Recherche S785, F-94800 Villejuif, France; and **Universite´ de Paris 10, Unite´ Mixte de Recherche S785, F-94800 Villejuif, France
1A.A. and F.B. contributed equally to this work.
Received for publication July 23, 2013. Accepted for publication October 6, 2013.
The work has been supported by grants from the Cance´ropoˆle Ile de France (CANC0701 and CACCR701), INSERM, and the Universite´ Pierre et Marie Curie, Paris, France. V.V. is a researcher from the Centre National de la Recherche Scientifique and the recipient of a Contrat Hospitalier de Recherche Translationnelle.
Address correspondence and reprint requests to Dr. Vincent Vieillard, INSERM Unite´ Mixte de Recherche S945, Laboratoire Immunite´ et Infections, Hoˆ pital La Pitie´Salpeˆtrie`re, 83 Boulevard de l’Hoˆpital, 75013 Paris, France. E-mail address: vincent. [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CT, cutaneous tumor; CTL, control; HCC, hepatocarcinoma; HD, healthy donor; KIR, killer Ig-like receptor; NCT, noncutaneous tumor; OLT, orthotopic liver transplantation; PTLD, posttransplant lymphoproliferative disorder.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

novo malignancies are the second major adverse outcome, after cardiovascular complications, with an incidence that ranges from 3 to 16% after OLT. Cancers cause 25% of the deaths following OLT, a rate significantly higher than in the general population (3–5). This increased risk was originally attributed, at least in part, to prolonged immunosuppressive treatments following transplantation (3).
As a part of innate immunity, NK cells are critical in protecting hosts from viral infections and tumor growth (6). An 11-y generalpopulation follow-up study showed that high NK cytotoxic activity is associated with reduced risk of cancer (7). In addition, NK cell infiltration into tumor tissues is associated with better disease prognosis in non–small-cell lung carcinoma (8), renal cell carcinoma (9), and colorectal carcinoma (10). NK cells are heterogeneous and differ in their proliferative potential, homing characteristics, functional capacities, and responses to different cytokines. They are divided into two major subsets: CD56bright and CD56dim cells, accounting, respectively, for 10 and 90% of the NK cells in the peripheral blood. On activation, CD56bright NK cells proliferate and produce cytokines; mostly IFN-g and/or TNF-a, but their cytotoxic activity is minimal. CD56dim NK cells, in contrast, proliferate minimally, produce relatively low amounts of cytokines, but are highly cytotoxic (11). It is not yet clear which NK cell subpopulation controls tumor growth most effectively. Recent experimental evidence demonstrates that NK development proceeds from a CD56bright to CD56dim phenotype (12, 13). NK cell function is regulated by a vast network of inhibitory and activating signals. Under normal immune surveillance, NK cells have inhibitory receptors that recognize MHC class I molecules as their cognate ligands; these receptors include killer Ig-like receptors (KIR)-L, LIR-1/ILT-2, LAIR-1, and the CD94/NKG2A heterodimer. Cytotoxicity occurs when stimulatory signals outweigh inhibitory ones by a critical threshold. Several of these activating receptors have been characterized, including NKG2C, NKG2D, and the natural cytotoxicity receptors (NKp30, NKp44, and NKp46) (14–17).

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Recent investigations have revealed that persistent viral infections influence NK cell phenotypes and functions. These include, in particular, the highly prevalent herpes viruses, such as CMV and EBV, which infect at least 50 and 80%, respectively, of the adult white population (18, 19). During latency, these viruses appear to be silent and cause no clinical symptoms as long as they are kept in balance with the host immune system. Rapid and vigorous proliferation of activated and differentiated CD56dimNKG2C+ NK cells in CMV-positive individuals was recently described upon additional encounters with viruses, including HIV-1, hantavirus, hepatitis B virus (HBV), and hepatitis C virus (HCV) (20–22), whereas the frequency of NK cells expressing NKG2C is low or undetectable in CMV-seronegative individuals (23). NKG2C recognizes HLA-E, although with a lower affinity than NKG2A does (24). In immunocompromised individuals, the reactivation of latent virus or the development of an acute CMV infection is associated with the promotion of a lasting increase in differentiated CD56dimNKG2C+ NK cells, as observed in an SCID child infected with CMV, but also after allogeneic transplantation or in acute CMV infection after organ transplantation (25–28).
Although the importance of NK cells in the control of CMV infection has been well documented, the implication of NK cell during CMV latency or reactivation after organ transplantation remains to be elucidated. The aim of this study was to determine the role of NK cells from liver-transplanted patients diagnosed with de novo solid malignancies. Our results suggest that CMV could drive the expansion of NKG2C+ NK cells associated with the development of specific tumors after OLT.
Materials and Methods
Patients and healthy donors
The characteristics of OLT patients are summarized in Table I. Samples were provided of patients at the time of diagnosis for their cutaneous tumors (CT; n = 18), or noncutaneous tumors (NCT; n = 24). OLT patients without de novo tumors and matched for sex and age were enrolled as controls (CTL) during the same period (n = 35). All patients came from either Paul Brousse (Villejuif, France) or Pitie´-Salpeˆtrie`re (Paris, France) hospitals and provided signed written informed consent. The appropriate institutional review board approved the study. Blood samples from 35 anonymous healthy donors (HD), provided by the hospital blood center (Etablissement Franc¸ais du Sang), served as CTL.
Cell lines
K562 (ATCC CCL-243) and P815 (ATCC TIB-64) were obtained from and validated by the American Type Culture Collection. The MHC class I– deficient 721-221 human cell line and transfected LCL-221-AEH cells, which express the E*0101 allele (22), were generously provided by D. Geraghty (Fred Hutchinson Cancer Research Center, Seattle, WA). All cell lines were maintained in RPMI 1640 medium supplemented with 10% FCS.
Flow cytometric analysis
NK cells were analyzed after staining with an appropriate Ab mixture, as previously described (13): anti-CD45 (Chrome Orange; #J33), anti-CD3 (ECD; #UCHT1), anti-CD56 (PC7; #N901), anti-CD159a/NKG2A (APC; #Z199), anti-CD336/NKp44 (PE; #Z231), anti-CD335/NKp46 (APC; #BAB281), anti-NKG2D (allophycocyanin; #ON72), anti-CD85j/ILT-2 (PE; #HP-F1), anti-CD69 (allophycocyanin; #FN50), anti–HLA-DR (PE; #Immu357), and anti-CD57 (FITC; #S-HCL-1) from Beckman Coulter; anti-CD161 (FITC; #DX12) and DNAM-1 (FITC; #DX11) from BD Biosciences; anti-CD337/NKp30 (PE; #AF29-4D12) and anti-KIR2DL2/ KIR2DL3/ (APC; #DX27) from Miltenyi Biotec; anti-NKG2C (PE; #134591), anti-KIR2DL1 (FITC; #143211), and anti-KIR3DL1 (allophycocyanin; #DX9) from R&D Systems; and anti-CD11b (AF700; #CBRM1/ 5), anti-CD11c (APC, #3.9), anti-CD62L (allophycocyanin eFluor780; #DREG-56), anti–integrin-a4 (PE; #9F10), and anti–integrin-b7 (FITC; #FIB504) from eBioscience.
For intracellular staining, PBMCs were fixed and permeabilized with 0.1% saponin in 0.5% BSA/PBS buffer and then stained with perforin (PE; #dG9; BD Biosciences), granzyme B (FITC; #GB1; BD Biosciences),

granzyme K (PE; #GM6C3; Santa Cruz Biotechnology), or granulyzin (AF647; #DH2; BD Biosciences), as described (13).
Populations of interest within the CD45+ lymphocyte population were gated on patterns of CD56/CD3 staining. Results are expressed as the percentage of positive cells within the gated population. At least 20,000 CD45+ cells were analyzed on a Navios cytometer (Beckman Coulter). Expression of each marker was measured as a percentage of the total CD32CD56+ NK cells. Hierarchical clustering was applied, and the results were displayed with the Genesis program (software available at http:// www.genome.tugraz.at), as previously described (29, 30).
Degranulation, intracellular cytokine production, and polyfunctional assays
Degranulation was assessed by the detection of CD107a on PBMCs cultured in the presence or absence of 1000 IU/ml proleukin-2 (Chiron) for 48 h. Briefly, PBMCs were incubated with K562, 721.211, or HLA-E–transfected 721.221 (221-AEH) target cells, at an E:T cell ratio of 1:1, in the presence of anti-CD107a mAb (FITC; #H4A3; BD Biosciences) (Supplemental Fig. 1A), as described (31). A redirected killing assay was performed against P815 target cells at a 1:1 E:T ratio. Cells were incubated in the presence of anti-CD107a and 10 mg/ml anti-NKG2C (#134591) or isotype control mAbs from R&D Systems (Supplemental Fig. 1B). After 1 h of incubation, Golgi Stop and Golgi Plug solutions (BD Biosciences) were added for an additional 5-h period. Cells were then stained with cell-surface markers (anti-CD3 and anti-CD56 mAbs), fixed, permeabilized with a Cytofix/ Cytoperm kit (BD Biosciences), and then stained with anti–IFN-g (Alexa Fluor-700; #B27; BD Biosciences) and anti–TNF-a (eFluor450; #Mab11; eBioscience) mAbs, as described (13, 31).
Polyfunctionality assays simultaneously detect several markers of NK cell functionality after these cells encounter target cells (31). Data were analyzed with FlowJo version 9 (Tree Star), and the rate of NK cells positive for zero, one, two, or three functions was defined with the software’s “Boolean gate” algorithm. Pestle software was used to remove the background, and pie charts, generated with Spice software (NIAI freeware) (32), present the frequency of NK cells positive for zero, one, two, or three responses (to CD107a, IFN-g, and TNF-a). Arcs depict the frequency of cells positive for CD107a, IFN-g, and/or TNF-a.
Statistical analysis
Statistical analyses were performed with Prism 5 software (GraphPad). Intergroup comparisons were assessed with the Fisher exact test for categorical variables, and the nonparametric Kruskal–Wallis test with Dunn posttest was used to define the significance of results from three independent groups of subjects, when compared 2 by 2. The nonparametric Mann– Whitney U test was used to compare two unrelated samples. Nonparametric correlations were assessed by determination of Spearman rank correlation coefficient. Statistical significance was defined by p , 0.05 with a two-tailed test: *p , 0.05, **p , 0.01, ***p , 0.001.
Results
Phenotypic signature of NK receptors in OLT patients developing de novo tumor
A total of 77 OLT patients (53 men and 24 women) were enrolled at the time of diagnosis, then categorized in function of the development of either de novo nonmelanoma CT or NCT, and compared with patients without tumors (CTL group). Table I summarizes their characteristics in detail; they received similar immunosuppressive treatments except for tacrolimus, which is reported to inhibit NK cell function, at least after renal transplantation (33) and was observed significantly more often in patients with CT (p , 0.0005). They did not differ significantly for the other variables considered, including sex, age, liver disease, age at transplantation, or follow-up post-OLT (Table I). Of 24 NCT patients who had de novo noncutaneous malignancies, five developed posttransplant lymphoproliferative disorders (PTLD); the other NCT included various genitourinary cancers (n = 7), colorectal cancers (n = 3), head/neck tumors (n = 3), various gynecological tumors (n = 3), lung cancer (n = 1), and hepatocarcinoma (n = 1) (Table II), as reported (3).
Flow cytometry was used to measure CD3+ T cells and CD32 CD56+ NK cells in OLT and HD. The absolute count and fre-

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Table I. OLT patient characteristics

CTL (n = 35)

CT (n = 18)

NCT (n = 24)

Sex, male/female (ratio) Age (y) Liver diseases
Cirrhosis [n (%)] HCC [n (%)] Others [n (%)] Transplantation age (y) Follow-up post-OLT (mo) Treatment post-OLT Azathioprine [n (%)] Mycophenolate [n (%)] Cyclosporine [n (%)] Tacrolimus [n (%)]

23/12 (0.66) 60 6 11
15 (43) 7 (20) 11 (31) 51 6 11 87 6 72
12 (34) 23 (66) 8 (23) 30 (86)

13/5 (0.72) 68 6 10
10 (56) 3 (17) 1 (5) 56 6 9 96 6 65
7 (39) 12 (67) 3 (17) 18 (100)

17/7 (0.71) 59 6 12
13 (54) 3 (13) 8 (33) 49 6 13 96 6 74
5 (24) 12 (57) 10 (48) 13 (62)

Statistical analysis was performed by *Fisher exact test or †ANOVA one-way test.

p Value
0.6202* 0.0632†
0.8746* 0.8621* 0.0691* 0.0732† 0.6966†
0.9436* 0.4122* 0.1578* 0.0005*

quency of lymphocytes subsets showed that OLT patients had significantly lower T cells than HD (Fig. 1A), whereas the distribution of NK cells was higher in OLT, in frequency and absolute count, irrespective of the presence of de novo tumors, as compared with HD (Fig. 1A). The analysis of NK cell subsets revealed that both CT and NCT displayed significantly higher levels of the CD56bright NK subset (mean 6 SD; CT 16.5 6 6.1%; NCT 14.1 6 5.5%; p , 0.0001 for both) than OLT CTL without tumors (10.5 6 5.7%) or HD (7.8 6 4.1%) (Fig. 1B).
We further compared the phenotypic properties of the NK cells in OLTs and show that NK cells from patients without de novo tumors were indistinguishable from those of HD, in terms of cellsurface expression of the major NK receptors studied (Fig. 1C, Supplemental Fig. 2). Importantly, the proportion of several markers was

significantly decreased in OLT patients with de novo tumors (CT and NCT), notably the expression of CD57, CD11b, and integrin-a4b7. In addition, as compared with the CT, the frequency of cells expressing NKG2D (p = 0.0018) and NKp30 (p = 0.0006), two activating receptors, was very significantly decreased in NCT, whereas CD62L increased (p = 0.042) (Mann–Whitney tests) (Fig. 1C). Overall, these results demonstrated that NK cells from OLT patients who developed de novo tumors display specific phenotypic NK cell features distinct from those of OLT CTL and HD.
Dichotomization of NCT patients by a hierarchical clustering of NK cell markers
To gain insight into the full characterization of NK cells in OLT patients with de novo tumors, we used a large-scale flow cytometric

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Table II. Individual characteristics of NCT patients

Patient No. Sex

Primary Disease

Graft Age (y)

Anti-CMV (IgG)

CMV Plasma (PCR)

Anti-EBV (IgG)

Active HBV (AgHBs)

2C2

92

M

Cirrhosis

55

2

96

M

HCC

52

+

192

F

Hepatitis

41

2

194

M

Cirrhosis

49

2

198

M Sclerosing

47

2

cholangitis

212

F

Cirrhosis

53

+

214

M

Cirrhosis

40

+

217

M

Cirrhosis

67

2

218

M

Cirrhosis

45

2

224

M

Cirrhosis

55

2

239

M

Cirrhosis

32

+

245

F

Cirrhosis

34

2

248

M

Cirrhosis

23

2

253

F

Cirrhosis

40

+

256

M Neuropathy

66

+

amyloidosis 2C+

89

M Hepatopathy

59

+

95

M Sclerosing

ND

+

cholangitis

195

F

Caroli

38

+

disease

206

F Neuropathy

40

+

amyloidosis

209

M

Cirrhosis

47

+

230

M

HCC

63

+

240

M

Hepatitis

51

+

261

F

Cirrhosis

ND

+

262

M

HCC

74

+

2

+

2

2

ND

+

2

2

ND

2

ND

2

2

2

2

2

ND

2

2

2

+

2

+

2

2

2

2

2

ND

2

2

+

+

2

2

2

2

+

2

2

+

2

2

+

2

2

2

ND

+

+

2

+

ND

2

2

+

2

+

+

2

+

+

2

2

+

2

+

+

+

+

+

2

F, female; Genitourinary, kidney or prostate; Gynecological, breast, uterine/ovary, or urothelial; M, male.

Active HCV
2 2 ND ND 2
2 2 2 2 2 + 2 2 + +
2 2
2
2
2 2 2 + 2

Time to Tumor (mo)

Tumors

11

Liver

98

Genitourinary

204

Genitourinary

231

Genitourinary

91

Genitourinary

234

PTLD

232

Genitourinary

62

PTLD

132

Genitourinary

98

Lung

89

PTLD

159

Gynecological

173

Genitourinary

35

Lung

121

PTLD

38

Head/neck

43

Colorectal

18

Gynecological

21

Colorectal

35

Colorectal

31

PTLD

85

Head/neck

116

Head/neck

25

Gynecological

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NK CELLS AND TUMORS POSTTRANSPLANTATION

FIGURE 1. Phenotypic features in
OLT patients. Flow-cytometric analysis was performed on lymphocytic CD45+
cells from HD and the three groups of
OLT patients: without tumors (CTL) or with de novo CT or NCT. (A) Frequency and absolute count of CD3+ T and CD32 CD56+ NK cells gated on the CD45+ lymphocytic gate. (B) Proportion of CD56bright cells among the CD32CD56+ NK cells. (C) Pattern of specific NK receptors determined among the CD32 CD56+ NK cell subset. Intergroup com-
parisons were assessed with Kruskal– Wallis test and Dunn posttest. *p , 0.05, **p , 0.001, ***p , 0.0001.

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analysis of the frequency of all 15 different phenotypic NK markers
used in this study (Fig. 2). This hierarchical clustering analysis
revealed specific clusters of patients relatively to the phenotypic
expression of NK markers; thus, OLT patients who developed CT
were similar to those in HD. In contrast, the NK cell repertoire of
patients with NCT was substantially different and can be divided into two particular groups called 2C2 and 2C+ (Fig. 2). Thus, 2C2 are mainly NKG2AhighCD62LhighILT2lowCD57lowNKG2C2, whereas the 2C+ was characterized by NKG2AlowCD62Llow ILT2highCD57highNKG2C+ NK cells. Notably, OLT CTL patients
without tumor represent an intermediate cluster between the HD/
NCT and the CT, with three individuals (CTL-91, 211, and 226) with an NK profile similar to 2C2 (Fig. 2).
To confirm these data, we further compared the phenotypic properties of some specific NK markers, both in CD56bright and CD56dim NK subsets, highlighted by the hierarchical analysis in the NCT patients of 2C2 and 2C+ NCT. Similar profiles were obtained in CD56bright cells, whereas in CD56dim cells, a higher proportion of NK cells expressed ILT2, CD57, and NKG2C in 2C+, as compared with 2C2, (Fig. 3A). In contrast, a high proportion of NK cells from 2C2 expressed NKG2A and CD62L, two
markers associated with an immature phenotype. These finding suggest that NK cells from 2C+ are highly more differentiated than those from 2C2. Accordingly, the percentage of NKG2C+ NK

cells was inversely correlated with NKG2A and CD62L and directly correlated with ILT2 and CD57 expression (Fig. 3B), as previously described (28, 34).
NKG2C expression skewed NK cell function in NCT patients
We next assessed the overall functional ability of NK cells. One of their main functions is the synthesis and release of cytokines, especially IFN-g and TNF-a (Supplemental Fig. 1A), which participate in triggering the adaptive immune response (35, 36). Fig. 4A shows that the level of intracellular IFN-g and TNF-a production was significantly different in 2C2 and 2C+. Thus, IFN-g production was almost undetectable in 2C+, but remained significantly higher (p = 0.012) in 2C2 than in CTL. In contrast, intracellular production of TNF-a was significantly upregulated in samples from 2C+ (p = 0.03), compared with 2C2 and CTL (Fig. 4A). Importantly, the percentage of NK cells expressing NKG2C was inversely correlated with their intracellular production of IFN-g (Spearman coefficient 20.76; p , 0.0001) and positively correlated with their TNF-a expression (Spearman coefficient 0.76; p = 0.0002) (Fig. 4B).
We further assessed the overall cytolytic activity of NK cells. Fig. 4C shows that granzyme B, perforin, and granulyzin were expressed at equivalent levels in all samples, whereas granzyme K, a marker of immaturity, increased only in NK cells from 2C2.

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FIGURE 2. Hierarchical clustering of the 15 combinations of NK cell markers from HD (black) and the three groups of OLT patients: without tumors (CTL; blue) or with de novo CT (green) or NCT (red). The percentage of each marker was measured independently on the CD32CD56dim NK cell subset.
Each column is dedicated to a definite NK marker. The color of each square reflects the percentage of expression of the corresponding marker in each
sample. The values measured for samples were color displayed and rank ordered considering the HD median as a reference: green color indicates inferior to median, and red color indicates superior to median. The groups 2C2 and 2C+ are indicated by dotted frames. Analysis was performed with the Genesis
program (available at http://www.genome.turgaz.at).

To obtain further insight into the function of NK cells, polyfunctional assays were performed in the presence of various target cells to determine simultaneously the levels of degranulation and cytokine production. As expected, in the absence of target cells, the level of CD107a was close to the baseline level in all study groups, but NK cells from 2C+ produced only TNF-a, whereas those from 2C2 produced mainly IFN-g (Fig. 5). In the presence of K562 or 721.221, two target cell lines that do not express MHC class I molecules, the proportion of polyfunctional cells is very low in 2C+ NCT, in which most TNF-a–positive cells are not cytotoxic. In contrast, the NK cells in the other groups had a greater tendency toward polyfunctionality with a high proportion of CD107+ cells producing cytokines (Fig. 5). In the presence of target cells expressing HLA-E (221.AEH), the ligand of the activating NKG2C receptor, and its inhibitory counterpart NKG2A, the degranulation

activity is strongly repressed in all groups, whereas their capacity to produce cytokines is preserved at the levels previously detected in the absence of targets (Fig. 5). Of note, a redirected killing assay against FcgR+ P815 target cells in the presence of anti-NKG2C mAb showed the continuing functionality of the activating NKG2C receptor expressed on NK cells of 2C+ NCT (Fig. 5, Supplemental 1A). Altogether, these data confirm that the inhibitory signaling mediated by NKG2A expression prevailed in all groups in the presence of HLA-E+ target cells, despite the expression of NKG2C in 2C+ NCT (Fig. 5), and suggest that NK cells from both NCT groups are mainly distinguished by their polyfunctional capacities.
Effect of CMV on NK cells in the development of de novo–specific malignancies in NCT patients
Finally, we sought to determine the contribution of these unique phenotypic and functional modulations of the NK repertoire in the

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FIGURE 3. Comparative expression of markers from 2C+ and 2C2 NCT
patients. (A) Box plots compare surface marker expression among CD32 CD56bright and CD56dim NK cells from 2C2 and 2C+ NCT. Intergroup compar-
isons were assessed with Mann–Whitney
tests. (B) Linear regression between
NKG2C expression and other marker from CD56dim NK cells of NCT patients. Open circles represent 2C2 and closed circles 2C+ NCT. **p , 0.001, ***p ,
0.0001.

development of specific malignancies. Table II revealed that 2C2 and 2C+ are different in regards to their primary disease; with a higher proportion of cirrhosis in 2C2 than in 2C+ (Fisher exact test, p , 0.03).
Intriguingly, all 2C+ patients (100%) are seropositive for CMV, compared with 26.6% (4 out of 15) of those in 2C2 (Fisher exact test, p , 0.0005), suggesting a striking association between 2C+ and CMV. Of note, these two groups did not differ significantly for
any of the other routinely tested viruses, including EBV, HBV, or
HCV (Table II). Moreover, quantitative PCR also detected CMV in 6 out of 9 2C+, compared with 0 out of 15 in 2C2 (Table II) and
1 out of 35 in control OLT patients without cancer (Supplemental
Table I). Although it is possible that the immunosuppressive
protocols used in OLT recipients could impair specific antiviral
immunity, NK cell function is apparently only sometimes affected
by these therapeutic regimens. Thus, pre- and postinduction treat-
ment did not differ significantly between both groups, although more patients of the 2C2 than 2C+ NCT (Fisher exact test, p =
0.1049) had cyclosporin in their postinduction treatment (Sup-
plemental Table II), consistent with previous data (37).
More importantly, Table II shows significant differences in
regards to the diagnosis of de novo malignancies in the two NCT
groups: colorectal (Fisher exact test, p = 0.0415) and head/neck (Fisher exact test, p = 0.0415) tumors appeared exclusively in 2C+,
whereas genitourinary (kidney or prostate) (Fisher exact test, p =

0.0223) tumors only appeared in 2C2. In contrast, gynecological (breast, uterine/ovary, or urothelial) tumors and PTLD were diagnosed in both NCT groups (Table II). These data suggest that phenotypic and functional features in NK cells from NCT, possibly associated with CMV, could drive the development of specific de novo malignancies.
Discussion
In this study of a cohort of 77 liver-transplanted recipients, we investigated the phenotypic and functional features of NK cells and their relation to the development of de novo cancers. Our results clearly show that NK cells from OLT with de novo tumors display an usual phenotype and impaired functioning. These changes are particularly evident in patients with NCT, and they may have the potential to affect the disease outcome. This highlights the possibility that unique NK cell subsets, closely associated with maturation defects and uncommon patterns of cytokine production, are expanded in NCT and may lead to the development of de novo tumors. Flow cytometric characterization of NK cells enabled us to distinguish two groups among the NCT patients: 2C2 is mainly composed of NKG2Ahigh CD62LhighCD57lowNKG2C2TNF-alowIFN-ghigh, with an immature signature, whereas 2C+ is characterized by nonconventional differentiated NKG2AlowCD62LlowCD57highNKG2C+TNF-ahigh IFN-glow NK cells.

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509

FIGURE 4. Intracellular cytokine production and expression of cytolytic markers in NK cells from 2C+ and 2C2 NCT patients. (A) Box plot plots summarize
intracellular production of IFN-g and TNF-a among CD3-CD56+ NK cells from 2C2 (n = 8) and 2C+ (n = 5)
NCT compared with HD (n = 10) and OLT patients
without tumors (CTL; n = 10). Intergroup comparisons
were assessed with Kruskal–Wallis test and Dunn posttest. (B) Linear regression between NKG2C ex-
pression and intracellular production of IFN-g or TNF-a. Open circles represent 2C2 and closed circles 2C+ NCT. (C) Expression of cytolytic markers among CD32CD56dim NK cells from 2C2 (n = 10) and 2C+ (n = 6) NCT compared with HD (n = 12) and OLT
patients without tumors (CTL; n = 12). Intergroup
comparisons were assessed with Kruskal–Wallis test and Dunn posttest. *p , 0.05, **p , 0.001.

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Our in-depth analysis reveals a striking link between the expansion of highly differentiated NK cells in 2C+ and CMV infection with all patients seropositive for CMV and a high proportion of CMV reactivation. Note that 60–90% of transplant recipients develop CMV infection due either to reactivation of a latent virus or to a new infection after long-term immunosuppressive treatments (37). CMV infection of tumor cells likely impairs NK cell function because CMV has evolved a unique strategy to circumvent its own virally mediated downregulation of MHC class I Ags to subvert NK cell recognition of infected cells (38). After intense debate and despite the difficulty of proving this experimentally, it is well accepted today that ∼20% of the global cancer burden is linked to infectious agents, including viruses, bacteria, and parasites (39). Although CMV has not yet been clearly implicated in human cancers, it has been detected in some malignant tumors, including colon cancer (40) and malignant glioblastoma with noncancer CMV-negative cells in close proximity to them (41). Recently, Baryawno et al. (42) showed significantly longer survival rates in medulloblastoma patients without CMV infection in the tumor than in those with high-grade infections. Interestingly, Di Cocco et al. (43) recently described a case of de novo gastric cancer after renal transplantation with a concomitant diagnosis of CMV disease.
Accumulating experimental evidence indicates that inflammatory cytokines, such as TNF-a, IFN-a, and GM-CSF, could be important in CMV-mediated tumors (44). In particular, TNF-a has

been linked to increased replication of CMV (45). In vitro, TNF-a directly stimulates the CMV immediate-early promoter and can dramatically increase malignant transformations by triggering the NF-kB transcriptional activator, thus contributing to the immune failure seen in various malignancies (46). Our experimental evidence supports this possibility: intracellular cytokine production in NK cells differed quite notably between both OLT groups with de novo tumors, with NK cells of 2C+ expressing mainly TNF-a and those of 2C2 produced more IFN-g. Popivanova et al. (47) showed that blocking TNF-a reduces carcinogenesis of this solid colorectal tumor, whereas IFN-g levels, as mediated by NK cells, predict long-term survival in patients with gastrointestinal stromal tumors after treatment with imatinib mesylate (48).
Our study underlines the complexity of NK cell responses and their clinical impact after liver transplantation. Our data confirm in CMV-negative patients the persistence of immature NK cells. In contrast, in CMV+ patients, a specific expansion of differentiated NK cells specific to the virus, as previously observed (21–23, 27, 28), partially biases the NK repertoire by a restriction of its capacities of the immune surveillance, as shown by a decrease of their polyfunctional capacities. This could favor the development of the specific malignancies, including colorectal and head/neck tumors, observed in 2C+. In contrast, the presence of immature NK cells in 2C2 leads to the development of de novo genitourinary tumors. Further investigations with larger numbers of patients are required to confirm these hypotheses; nonetheless, the findings that

510

NK CELLS AND TUMORS POSTTRANSPLANTATION

Coordinators for pharmacogenetics: Laurent Puig,8 Laurent Thervet8 Coordinators for histology: Anne Janin,1 Martine Raphae¨l11 1Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Saint-Louis, Paris, France. 2Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Henri Mondor, Cre´teil, France. 3Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France. 4Hoˆpital Foch, Suresnes, France. 5Hoˆpital Marie Lannelongue, Le Plessis-robinson, France. 6Hoˆpital Saint-Joseph, Paris, France. 7Assistance
Publique-Hoˆpitaux de Paris, Hoˆpital Cochin - Saint Vincent de Paul, Paris, France. 8Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Europe´en Georges Pompidou, Paris, France. 9Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Bichat, Paris, France. 10Assistance Publique-Hoˆ pitaux de Paris, Hoˆ pital Necker - Enfants Malades, Paris, France. 11Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Biceˆtre, Le Kremlin-Biceˆtre, France. 12Assistance PubliqueHoˆpitaux de Paris, Hoˆpital Tenon, Paris, France. 13Assistance PubliqueHoˆpitaux de Paris, Hoˆpital Paul Brousse, Villejuif, France. 14Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Saint-Antoine, Paris, France. 15Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Beaujon, Clichy, France. 16INSERM,
UMR-S 945, Paris, France.

Disclosures
The authors have no financial conflicts of interest.

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FIGURE 5. Polyfunctionality of NK cells from NCT patients. Polyfunctionality assays of CD32CD56+ NK cells from 2C2 (median; n = 5), and 2C+ (median; n = 5) NCT compared with HD (median, n = 5) and OLT patients without tumors (CTL; median; n = 12). Cell-surface expression of CD107a and intracellular production of IFN-g and TNF-a were assessed in absence of target cells (None) or in the presence of K562, 721.221 that did (221.AEH) or did not (721.221) express HLA-E, and P815 6 anti-NKG2C mAb (aNKG2C). FlowJo software and its Boolean gate function were used to determine whether cells expressed zero, one, two, or three functions simultaneously. Data represent the mean of frequency of five independent samples and are presented as pie charts representing the number of different responses and colored arcs the frequency of cells producing each cytokine, created with Pestle and Spice software.
several specific cancer forms are linked to a possible association between the NK cell maturation status and/or the patient’s CMV status could open up new possibilities for neutralizing development of certain NCT after OLT.
Acknowledgments
We thank all of the clinicians and patients who participated in the K-GREF multicenter study. We also thank Christophe Parizot (De´partement d’Immunologie, Hoˆpital La Pitie´-Salpeˆtrie`re, Paris, France) for excellent technical support and helpful discussion.
K-GREF Study Group
Principal coordinator: Michel Marty1 Coordinators for the clinical and biological registries: Daniel Azouley,2 Marc-Olivier Bitker,3 Alain Chapelier,4 Jean-Paul Couetil,2 Philippe Dartevelle,5 Michel Delahousse,4 Philippe Deleuze,6 Bertrand Dousset,7 Denis Glotz,1 Laurent Hannoun,3 Philippe Lang,2 Francoise Le Pimpec Barthes,8 Patrick Nataf,9 Patrick Niaudet,10 Jean-Jacques Patard,11 Alain Pavie,3 Eric Rondeau,12 Didier Samuel,13 Emmanuel Tiret,14 Dominique Valla,15 Coordinator for epidemiology: Caroline Besson11 Coordinators for immunology: Patrice Debre´,16 Antoine Toubert,1 and Vincent Vieillard16

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