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Human Immunology 72 (2011) 229–237

Contents lists available at ScienceDirect

Divergences in KIR2D� natural killer and KIR2D�CD8� T-cell reconstitutionollowing liver transplantationM. R. LÔpez-Alvarez a,d,†, J. A. Campillo a,d,†, I. Legaz a, R. M. Blanco-GarcÎa a, G. Salgado-Cecilia a,. M. BolarÎn a, L. Gimeno a, J. Gil b, A. M. GarcÎa-Alonso a,d, M. Muro a,d, M. R. Alvarez-LÔpez a,d,. Miras c,d, A. Minguela a,d,*

a Immunology Service, University Hospital Virgen de la Arrixaca, El Palmar, Murcia, Spainb Immunology Service, University Hospital Gregorio, Maraðòn, Madrid, Spainc Digestive Medicine Service, University Hospital Virgen de la Arrixaca, El Palmar, Murcia, Spaind Centro de Investigaciòn Biomèdica en Red de enfermedades hepàticas y digestivas (CIBERehd)

A R T I C L E I N F O

Article history:Received 6 September 2010Accepted 27 December 2010Available online 4 January 2011

Keywords:NK cellsCD8 T lymphocytesKIR2D receptorsLiver transplantationTolerance

A B S T R A C T

Natural killer (NK) and CD8� T cellsmay be active elements in the allograft response, but little is known abouttheir role in liver transplantation. Some of these cells express killer immunoglobulin-like receptors (KIRs),which after binding specific ligands may transmit inhibitory/activating signals. In this study, circulating NKand CD8� T cells expressing CD158a/h (KIR2DL1/S1) or CD158b/j (KIR2DL2/3/S2) receptors were analyzed in142 liver recipients by flow cytometry. They were underrepresented in patients before transplantation, butfollowing transplantation, whereas the KIR2D� NK subsets experienced a late recuperation (day 365)mainlyin C2-homozygous patients developing early acute rejection, recovery of the 2 CD8�KIR2D� T cells startedearlier, showing significant differences on day 365 between patients without acute rejection and thosesuffering from it (p � 0.004 and p � 0.0001, respectively). These differences were also evident when thehuman leukocute antigen-C genotypes of the recipient were considered. In conclusion, whereas the laterecovery of KIR2D� NK cells in C2/C2 patients appears to be linked to acute rejection, the increase in earlyCD8�KIR2D� T cells in overall liver recipients correlates with a most successful early graft outcome. There-fore, monitoring of KIR2D� cells appears to be a useful tool for liver transplant follow-up.

� 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights

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1. Introduction

Liver allografts are well accepted in the absence of human leu-kocyte antigen (HLA) matching [1–3], and despite the recentlysuggested involvement of the HLA-C locus in long- and short-termtransplant outcome [4,5], its role is still controversial [6]. In addi-tion, other recent studies point to a contribution of natural killer(NK) cells rather than CD8� T effector cells in early acute rejection[7–13].

Human NK cells are crucial components of the innate immunitythat are able to lyse infected and tumor target cells without priorsensitization [14–16]. The NK cell function is linked to lectin-likeCD94:NKG2 and killer immunoglobulin-like receptors (KIR), bothof which are expressed in a clonally distributed mode [17–19].After interaction with HLA class I specific ligands on target cells,these receptors transmit a balance of positive and negative signalsthat ultimately regulate the NK cell function [20–22]. KIR recep-

* Corresponding author.E-mail address: alfredo.minguela@carm.es (A. Minguela).

´

† The contribution ofM.R. LÔpez-Alvarez and JA. Campillo is equal and the order ofauthorship is arbitrary.

0198-8859/11/$32.00 - see front matter � 2011 American Society for Histocompatibilitydoi:10.1016/j.humimm.2010.12.015

tors, originally described onNK cells,may also be detected inminorperipheral blood CD8� T, CD4� T, or �� T-cell subsets [21,23,24].

In humans, KIR receptors can harbor 2 (KIR2D) or 3 (KIR3D)xtracellular immunoglobulin (Ig)-like domains and recognize dif-erent groups of HLA class I alleles that are determined by themino acids at position 80 of the �1-helix [25,26]. In particular,

inhibitory KIR2DL1 and activating KIR2DS1 receptors recognizeHLA-Cmolecules exhibiting a lysine residue at position 80,whereasinhibitory KIR2DL2/3 and activating KIR2DS2 receptors interactwith HLA-C molecules bearing an asparagine in that position[21,27]. Importantly, the functional competence of NK cells can beacquired through the interaction of KIR receptors with cognateclass I ligands [22,27,28].

Several studies have investigated the NK cells in human hema-opoietic stem cell transplantation (HSCT) and others in organransplants animal models [29–33], but much less is known aboutheir involvement in liver transplantation [8]. However, there areew studies regarding the peripheral blood lymphocytes that ex-ress KIR receptors in organ transplantation [5,34,35]. Therefore,he intent of this work was to study both NK and CD8� T-cell

populations expressing KIR2D receptors before and following liver

and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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transplantation to ascertain their possible connection with theearly transplant outcome.

2. Subjects and methods

2.1. Studied cohorts

A series of 142 consecutive liver transplants were recruitedfrom2 Spanish Centers, Virgen de la Arrixaca University Hospital inMurcia and Gregorio MaraÒÔn University Hospital in Madrid, tak-ing part in a transplant immunology network and sharing studyprotocols. Thedemographic data of patients and clinical indicationsare summarized in Table 1.

Additionally, a series of 46 sex- and age-matched healthy Cau-asian individuals were also studied. In all cases, informed consentn writing was obtained, and the institutional ethical committeepproved the protocol of the study, which conformed to ethicaluidelines.

.2. Immunosuppression and acute rejection diagnosis

Prophylactic immunosuppression consisted of standard drugherapy with cyclosporin A or tacrolimus in the presence or ab-ence of mycophenolate and methylprednisolone. All patients re-eived a similar initial dose of the standard therapy that was sub-equently adjusted to maintain the recommended level for eachmmunosuppressant. Acute rejection (AR) episodes were treatedith high doses ofmethylprednisolone (bolus of 1 g) for 3 days, andnce AR reversed, all patients followed a similar treatment. ARiagnosis was based on clinical, biochemical, and histologic crite-ia, considering a triad of features: (1) mixed portal inflammation,2) billiard epithelium or intrahepatic bile duct injury, and (3)ubendothelial inflammation of portal and terminal hepatic veinranches [36].

.3. Viral preinfection diagnosis

Viral hepatitis C virus (HCV) or cytomegalovirus (CMV) pre-nfections were determined by measuring serum-specific anti-odies. In the case of HCV, a qualitative immunoassay (AxSYMHCV3.0, Abbott, Wiesbaden-Delkenheim, Germany) was used, and

he results were confirmed by quantitative reverse transcription-olymerase chain reaction (REAL, C.E. Durviz, Valencia, Spain). IgGnti-CMV-specific antibodies were tested by a quantitative chemi-uminescence immunoassay technique (Liason CMV IgG, DiaSorin,taly). Hepatitis B was tested by a qualitative immunoassay (HBsAgI, cobas, Germany) and also confirmed by reverse transcription-olymerase chain reaction (REAL, C.E. Durviz).

.4. Protocol design and flow cytometry

Blood samples were collected by venous punction on day 0pretransplant) and on days 7, 15, 30, 90, and 365 (1 year) following

Table 1Demographic data and indications for transplant in total liver recipients

Gender n (%)

GenderMale 102 (72)Female 40 (28)ean age (years) 53 � 1retransplant viral infectionHCV 35 (61)HBV 14 (25)CMV 5 (9)Others 3 (5)

BV, hepatitis B virus; HCV, hepatitis C virus; CMV, cytomegalovirus.

ransplantation, according to a consensus protocol between partic-pating institutions.

Monoclonal antibodies recognizing CD45 (2D1) and CD8 (SK1)erCP-conjugated, CD3 (SK7) and CD14 (MAP9) APC-conjugated,nd PE-CD19 (4G7) were provided by Becton–Dickinson (BD, Sanose, CA). FITC-CD28 (CD28.2), PE-CD158a/h (EB6), and PE-D158b1/b2/j (GL183) recognizing KIR2DL1/S1 and KIR2DL2/L3/2, respectively, were from Beckman Coulter (Fullerton, CA). Iso-ype-matched antibodies were used to set the fluorescenceackground. Fresh peripheral blood cellswere stained using appro-riate combinations of these monoclonal antibodies [37].Labeled cells were acquired in a FACScalibur flow cytometer by

ellQuest software (BD), and the analysis was carried out usingaint-A-Gate software (BD). A “lymphocyte gate”was definedusingorward/side scatter settings corresponding to a cell populationxpressing �96% CD45 and �1% CD14. As previously reported [38],

within this gate, KIR2D� NK cells were taken to be those negativeor both CD3 and CD19 expressing CD158a/h or CD158b1/b2/j mol-cules (KIR2DL1/S1 or KIR2DL2/3/S2 receptors, respectively),

whereas the KIR2D-positive CD3�CD8�CD28- T cells were thoselso expressing CD158a/h or CD158b1/b2/j molecules, hereaftereferred to as CD8�KIR2D� T cells,

.5. HLA-C genotyping

HLA-C genotyping was performed by a Dynal RELI SSO HLA-Cyping kit (Dynal Biotech ASA, Oslo, Norway), which permitted theLA-C dimorphism at position 80 of the �1-helix to be distin-uished. The HLA-C alleles were assigned to the 2major KIR-ligandroups, C1 and C2, as previously reported [39,40].

.6. Statistical analysis

Data were collected in a database (Microsoft Access 2.0; Mi-rosoft Corp., Seattle, WA), and statistical analysis was performedsing SPSS 15.0 software (SPSS, Inc., Chicago, IL). Comparisonsetween groups of patientsweremadeusing the two-sided Fisher’sxact test. Parametric unpaired two-tailed Student t or nonpara-etric Mann–Whitney tests were used to compare mean values ofIR2D� NK or CD8�KIR2D� T cells. Additionally, the parametric

paired two-tailed t or the nonparametric paired Wilcoxon testswere applied to compare results at different posttransplant times.All data were expressed as means � SEM, and the level of signifi-cance was set at p � 0.05.

3. Results

3.1. Clinical characteristics of liver recipients

In this series, ARwas histologically demonstrated in 20% of livertransplants, of which 86% occurred within the first month aftertransplantation (respectively, 7, 14, and 3 cases in the second, third,

Indications for transplant n (%)

Alcoholic cirrhosis 61 (43)Viral chronic hepatitis 37 (26)Hepatitis C 22Hepatitis B 9Non C non B 6

Hepatocarcinoma 13 (9)Autoimmune diseases 11 (8)Alcoholic cirrhosis plus HVB and/or HCV 7 (5)Cryptogenic cirrhosis 5 (4)Fulminant hepatitis 2 (1)Others 6 (4)

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M. R. Lòpez-Alvarez et al. / Human Immunology 72 (2011) 229–237 231

and fourth posttransplant weeks). Of the remaining cases of rejec-tion (14%), 1 appeared in the second month and 3 in the thirdmonth. In patients with alcoholic cirrhosis or HCV preinfection, theAR ratewas similar (21.3 and 20%, respectively). No associationwasfound between acute rejection and age, gender, or prevalentpathologies.

For the analysis, patients were classified according to the pres-ence or absence of acute rejection into 2 groups, nonacute rejection(NAR; n � 114) and AR (n � 28), and according to the HLA-Cllotypes as C1/C1, C2/C2 homozygous or C1/C2 heterozygousatients.

.2. Analysis of leukocyte populations before transplantation

Total leukocyte and lymphocyte counts obtained in liver recip-ents before and following liver transplantation are summarized inig. 1. Before transplantation, the leukocyte number did not differetweenpatient and control groups or betweenNAR andARgroups

Fig. 1. Number of leukocytes and lymphocytes in peripheral blood from liver recrespectively, at pretransplant time in controls, nonacute rejection (NAR) patients, anand AR patients, respectively. (E) shows the changes of total T lymphocytes (left),

uring the follow-upperiod. Controls (open triangles), NAR (open squares), andAR (solid sqf normal individuals. Significant values with respect to day 0 are represented as *p � 0.0

of patients (Fig. 1A), although the mean number of total lympho-cytes was significantly lower in the 2 groups of patients than inhealthy individuals (Fig. 1B; p � 0.0001), thus evidencing lym-phopenia in patientswaiting for a liver transplant, compensated byan increase in monocytes (data not shown).

3.3. Evolution of leukocyte populations during the firstyear posttransplant

The posttransplant follow-up revealed that mean values of leu-kocytes were always close to those of the healthy individuals,except on days 7 and 15 after transplantation (Fig. 1C); on thesedates the leukocytes of the 2 groups of patients significantly out-numbered those of the controls (p � 0.01).

With regard to lymphocyte evolution, their total mean numberremained below normal values at all times (Fig. 1D) and graduallyincreased in NAR patients from day 7 onward, reaching significantdifferences on day 365 with respect to the mean basal value (p �

s and controls. (A) and (B) illustrate values of leukocytes and total lymphocytes,atients. (C) and (D) represent evolution of leukocytes and total lymphocytes in NART lymphocytes (middle), and CD8� T lymphocytes (right) in NAR and AR patients

ipientd AR pCD4�

uares). Data are expressed asmeans� SEM.Dotted lines represent themean values1, **p � 0.05, and ***p � 0.005.

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M. R. Lòpez-Alvarez et al. / Human Immunology 72 (2011) 229–237232

0.013). After day 90, the number of total T lymphocytes signifi-cantly increased in concomitancewith CD8� T cells (Fig. 1E, left andright, respectively; p� 0.005),whereas therewas no clear evidenceof a simultaneous increase in CD4� T lymphocytes (Fig. 1E,middle).Therefore, the recovery of total T lymphocytes appears to bemainlyrelated to that of CD8� T cells. In contrast, as shown in Table 2, thetotal number of NK cells experienced only slight variations and thenumber of B lymphocytes had decreased at the end point.

3.4. Peripheral blood NK and CD8+ T cells expressing KIR2D in liverecipients before transplantation and in healthy individuals

The results of this analysis indicated that the percentages ofoth total NK andCD8� T cells in thewhole series of liver recipients,

as well as in NAR or AR patients (Table 3), did not differ from that ofthe control population (p � 0.05). In contrast, the percentages ofCD158a/h� and CD158b/j� NK cell subsets were significantly lowern patients than in controls (p � 0.0001). This reduction in fre-uency was also observed in both the NAR and the AR groups (p �.0001, except for CD158b/j� NK cells in the AR group, p � 0.02).ith regard to the CD8�KIR2D� T lymphocyte populations, theD8�CD158b/j� T cells proportion decreased with respect toealthy individuals only in AR patients (p � 0.02).In terms of mean absolute values, a significant reduction of the

otal NK and CD8� T-cell number was found in the patients (p �0.0001 and p � 0.001, respectively). Among the populations ex-pressing KIR2D, both KIR2D� NK cell subsets were significantlynderrepresented in patients with respect to healthy individualsp � 0.0001), whereas no changes were seen in the CD8�KIR2D� Tymphocyte subsets. Furthermore, the absolute number of KIR2D�

NK, but not that of CD8�KIR2D� T cells, was significantly reduced inboth NAR and AR patients with respect to that of the controls (p �0.0001).

Table 2Absolute number of total NK cells and B lymphocytes in liver recipients accordingto clinical and histologic characteristics during the follow-up period

Day NK cells(cells � 10�3/mL)

B lymphocytes(cells � 10�3/mL)

NAR AR NAR AR

0 231 � 21 159 � 22 118 � 11 129 � 247 116 � 10 95 � 14 157 � 14 158 � 33

15 145 � 15 115 � 20 142 � 16 169 � 3830 142 � 13 115 � 11 157 � 20 175 � 3490 172 � 18 110 � 18 128 � 20 111 � 2565 247 � 31 181 � 67 81 � 9 43 � 15

AR, non-acute rejection; AR, acute rejection. Cell numbers representmeans� SEM.

able 3ercentage and absolute number of peripheral blood NK and CD8� T cells expressin

Controls (n � 46) Liver recipients

% Cells � 10�3/mL Total (n � 142)

% Cells � 10�3

K cellsTotal NK 20 � 1 341 � 22 22 � 2 217 � 17b

CD158a� 16 � 2 52 � 7 8 � 1a 17 � 2b

CD158b� 26 � 2 80 � 9 13 � 2a 28 � 4b

CD8� T cellsTotalCD8� T

21 � 1 390 � 31 25 � 1 265 � 19d

CD158a� 4 � 1 8 � 2 3 � 1 6 � 1CD158b� 13 � 2 22 � 5 9 � 1 21 � 2

a,bp � 0.0001, cp � 0.02, and dp � 0.001 indicate differences between controls and lercentages of total NK and CD8� T cells were calculated with respect to total lym

�

respect to total NK or CD8 T cells; cell numbers represent means � SEM. p values were dNAR, non-acute rejection; AR, acute rejection.

3.5. Evolution of NK KIR2D+ and CD8+ KIR2D+ T cells in NAR and ARpatients along the post-transplant follow-up

Following liver transplantation and independent of the immunesuppressive treatment, KIR2D� NK and CD8� T-cell patternsvolved in a divergent manner (Fig. 2). Thus, percentages ofD158a/h� and CD158b/j� NK cells did not significantly differ be-ween NAR and AR patients (Fig. 2A and B), and their absolutealues only exceeded those of day 0 or day 365 (Fig. 2C and D).onetheless, these differences were only significant in the NARroup on day 365 (p � 0.021 and p � 0.027, respectively, forD158a/h� and CD158b/j� NK cells). Of note, although the cellumber of KIR2D� NK subtypes was slightly higher in AR than in

NAR patients, differences were not statistically significant (p �

0.05).The percentages of CD8�CD158a/h� and CD8�CD158b/j� T lym-

phocytes were always higher in NAR than in AR patients on days 7and 15 after transplantation, when the differences reached statis-tical significance (Fig. 2E and F), particularly those of theCD8�CD158a/h� T lymphocytes on day 90 (Fig. 2E). In addition andmportantly, both in NAR and in AR patients, a full recovery of thebsolute number of CD8�CD158a/h� and CD8�CD158b/j� T cells

took place by day 30 after transplantation. Subsequently, only inthe NAR group, these populations gradually increased fromdays 30to 365, reaching significant differences with respect to pretrans-plant values on days 90 and 365 (p � 0.005 and p � 0.0001,espectively, for CD158a/h� and CD158b/j� cell subsets). In con-rast, in AR patients, although these cells surpassed basal levelsromday 30 onward, their increaseswere almost negligible. Hence,n day 365, absolute values of both types of CD8�KIR2D� T cell

subsets were significantly lower in AR than in NAR patients (p �

0.004 and p � 0.0001, respectively, for each subset).

3.6. KIR2D+ NK and CD8+ T-cell reconstitution in recipients withifferent HLA-C genotypes

As shown in Fig. 3, KIR2D-positiveNK andCD8� T cellswere alsoexamined, taking into consideration the different HLA-C genotypesof the recipient. As shown in columns A and C of Fig. 3, mean valuesof KIR2D� NK cell subsets increased in C2-homozygous patients athe end of the follow-up period (day 365), the time in which signif-cant differences were observed between patients experiencing orot experiencing early AR episodes (AR vs NAR groups; p � 0.012or CD158a/h and p � 0.05 for CD158b/j). Conversely, these cellubsets remained at basal levels during follow-up in patients bear-ng the C1/C1 genotype, and only the CD158b/j� NK cell subsetsignificantly increased in C1/C2 heterozygous patients who exhib-ited a good early graft acceptance (p � 0.05).

2D receptors in healthy controls and in pretransplant liver recipients

NAR (n � 114) AR (n � 28)

% Cells � 10�3/mL % Cells � 10�3/mL

23 � 1 231 � 21b 19 � 2 159 � 22b

7 � 1a 16 � 2b 9 � 2a 19 � 7b

12 � 2a 29 � 4b 13 � 2c 24 � 5b

24 � 1 267 � 20d 28 � 3 258 � 55d

3 � 1 6 � 1 2 � 1 7 � 29 � 1 22 � 3 7 � 1a 17 � 4

cipients.tes; percentages of cells positive for CD158a/h and CD158b/j were calculated with

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M. R. Lòpez-Alvarez et al. / Human Immunology 72 (2011) 229–237 233

Fig. 2. Peripheral blood NK and CD8 T-cells positive for CD158a/h and CD158b/j from overall liver recipients. (A–D) summarize the percentages or absolute values of NKexpressing CD158a/h and CD158b/j in NAR and AR patients during the follow-up period. (E–H) show percentages or cell numbers of CD8� T lymphocytes that express

D158a/h and CD158b/j in both NAR and AR patients. Data are expressed asmeans � SEM, NAR (open bars/squares), and AR (solid bars/squares). Dotted lines representmeanalues of normal individuals. *p � 0.05, **p � 0.005, and ***p � 0.0001 express the level of significance with respect to day 0.

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M. R. Lòpez-Alvarez et al. / Human Immunology 72 (2011) 229–237234

Concerning the CD8�KIR2D� T lymphocyte subpopulations (Fig.3, columns B andD), an earlier and significant increasewas found inboth C1 and C2 homozygous recipients only when AR was absent(NAR group). In C1/C2 recipients CD8�CD158a/h� T lymphocytesncreased after day 90 (p � 0.01), whereas the behavior of theD8�CD158b/j� subset resembled that observed in C1 and C2 ho-ozygous patients.

.7. Posttransplant evolution of KIR2D+ NK and CD8+ T cells in liverrecipients in whom transplant indication was alcoholic cirrhosis orHCV preinfection

The potential influence of these representative pathologieson the KIR2D� cells recovery was also assessed in the NAR andAR groups of patients (Fig. 4). Thus, in patients suffering fromalcoholic cirrhosis, the evolution of NK and CD8� T cells express-ng CD158a/h and CD158b/j molecules closely resembled thatbserved when overall transplant indications were examined aswhole (Fig. 4A–D).However, when HCV preinfected patients were independently

onsidered, the analysis of both KIR2D� NK cell subsets (Fig. 4E and) revealed that only NAR patients, but not AR patients, had recov-red basal values by the end of the follow-up period (day 365).oreover, although the counts of CD8�KIR2D� T lymphocytes inCV preinfected patients were the highest, their progression wasimilar to that seen in overall patients or in those suffering fromlcoholic cirrhosis, and no statistical differenceswith respect to the

Fig. 3. Recuperation ofNKandCD8T cells expressingCD158a/h or CD158b/j receptorD158a/h or CD158b/j receptors in NAR (open squares) and AR (solid squares) whenD158a/h� NKand CD8� T cells, and (C) and (D) show changes in both populations poines represent the mean values of normal individuals. Data represent means � SEM

ther pathologies were detected (Fig. 4G and H).

4. Discussion

The findings of this study provide evidence of a divergence inthe recuperation of peripheral blood NK and CD8� T cells express-ng KIR2D receptors following liver transplantation. Such diver-ence was discernible in the proportion, the absolute number, andhe kinetics exhibited by these cells either in the total number ofecipients who experienced or did not experience early AR or inhose recipients bearing different HLA-C allotypes.

Effectively, the recovery of NK cells expressing KIR2DL1/S1 orIR2DL2/3/S2 took place only at the end of the follow-up period inatients developing AR, whereas that of the CD8�CD28- T lympho-

cytes that expressed any of these KIR2D receptors arose earlier inall patients exhibiting a good early graft acceptance. The detectionof a low number of circulating total NK cells and KIR2D� NK cellsubsets (including KIR2DL1/S1 or KIR2DL2/3/S2)encompassing theeriod of highest AR risk is in accordancewith prior studies report-ng an absence of correlation between NK cell activity or a reduc-ion in circulating KIR2D�NKcells andAR [8,35]. Indeed, in the casef C1 homozygous patients, both KIR2D� NK cell subsets remained

at reduced levels at all time points, but not in those lacking the C1ligands (C2/C2 homozygous), in whom these NK subsets showed alate recuperation, particularly in patients displaying AR episodes.

By contrast, the NK cell profiles contrasted with those of theCD8�KIR2D� T lymphocytes, because percentages of these T-lym-phocyte subsets were prematurely increased when patients were

cipientswith differentHLA-C genotypes. Changes ofNKandCD8�T cells expressingLA-C genotype of patients was considered. Columns (A) and (B) illustrate values offor CD158b/j for C1/C1 (n� 55), C1/C2 (n� 66), and C2/C2 (n� 21) patients. Dotted*p � 0.01, **p � 0.001, and ***p � 0.05 indicate the different levels of significance.

s in rethe H

free of AR. The bias of KIR2D�NKcell number observed at the endof

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M. R. Lòpez-Alvarez et al. / Human Immunology 72 (2011) 229–237 235

the follow-up period in C2/C2 homozygous patients experiencingAR also contrastedwith the higher CD8�KIR2D� T-cell expansion inpatients without AR independent of the HLA-C genotype. Alto-gether, differences in theKIR2D�NKandT cells, at least in part,maybe responsible for the differences in the frequency of AR previouslydescribed in C1 and C2 homozygous recipients [5]. Thus, our data

Fig. 4. CD158a/h� and CD158b/j� NK or CD8� T cells in liver recipients with alcoholhereas (C) and (D) illustrate the values of CD158a/h� and CD158b/j� CD8� T cells iD158b/j� NK cells or CD158a/h� and CD158b/j�CD8� T cells in HCV preinfected pa

for each time. Dotted lines represent the mean values of normal individuals. *p � 0

suggest that in liver recipients, the recuperation of KIR2D� NK cells

would be subrogated to the concomitant presence of C2 ligands andearly AR, whereas that of CD8�KIR2D� T cells appear to be lessrestricted to C-ligands and linked to a better early liver graftsuccess.

This KIR2D� cell skewing is difficult to explain, but 2 majorhypotheses implying concurrent mechanisms could be envisaged:

osis or HCV preinfection. (A) and (B) show values of CD158a/h� and CD158b/j� NK,ents suffering from alcoholic cirrhosis. (E–H) show the evolution of CD158a/h� and. NAR (open squares) and AR (solid squares). Data are represented as means � SEMd **p � 0.05 indicate the different levels of significance.

ic cirrhn pati

(1) NK cells, as effectors of the innate immunity, would quickly

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M. R. Lòpez-Alvarez et al. / Human Immunology 72 (2011) 229–237236

target the graft and be removed from the circulation, and (2) themajor capability of T lymphocytes to proliferate against chronicallogenic stimuli could support the earlier enhancement of circu-lating CD8�KIR2D� T lymphocytes. Taking this view, early alloacti-ated NK cells could be confined within the liver, mainly acting inhe earlier alloresponse, which leads to AR, as proposed in animalodels, where NK cells rather than CD8� T lymphocytes have been

considered the major factors infiltrating the liver [13,41].The processes that govern the size of KIR2D� NK cell subsets

emain to be determined. In effect, the likelihood of the C-ligandediation has also been considered [42], but a recent study stated

hat autologous HLA-C genotypes do not condition KIR2DL1 orIR2DS1 NK cell emergence [43]. In this line, data presented herend compelling evidence fromHSCT and liver transplantation pointo HLA-C ligands or genotypes playing some role in shaping KIR2Dells in allogenic situations [4,5,30]. Furthermore, in accordanceith the findings of Zenhaeusern et al. [44] regarding the interleu-in 10-producing NK cells with potential regulatory ability in-uced in vitro, the late in vivo emergence of KIR2D� NK cells in

C2/C2 patients experiencing early AR could derive into NK regu-latory cells able to counteract the potent alloreactive challengelinked to early AR.

Alternatively, in agreement with our preliminarily report, inwhich the follow-up of liver recipients was only performed duringthe first posttransplant month [35], the early and remarkable aug-ment in the proportion of CD8�KIR2D� T lymphocyte in patientsho did not develop AR is related to a substantial chance foruccessful early engraftment. This finding is consistent with theeported decrease of CD158b� T cells in kidney transplants experi-ncing AR [34].The intrinsic mechanisms allowing the KIR2D expression on T

ells are not well understood, but KIR receptors are witness to aD8� T cell advanced state of differentiationwith reduced capacityo proliferate upon T-cell receptor engagement of conventionalntigens [18,45,46]. Nonetheless, the strength of the chronic chal-enge hold by graft alloantigens could have a direct impact on theD8�KIR2D� T-lymphocyte amplification from the earlier timeoints. The question is, why do CD8�KIR2D� T cells become more

highly represented in patients achieving less early graft injury?Oneoption is that, as proposed for tumors [45], the alloreactivity ofthese cells may be inhibited after rearrangement at the T-cell re-ceptor locus through the binding of their KIR with cognate C-ligands, and the other is that CD8�KIR2D� T cellsmight exert a trueactive regulatory action by the elimination of bystander alloreac-tive T lymphocytes lacking specific C-ligands.

It is important to note that the increases in CD8�KIR2D� T-cellpatterns following liver transplantation appear independent onconcomitant leukocytosis during the first 2 posttransplant weeks(maybe because of the transplant-associated inflammatory pro-cess), given that at these times the total lymphocyte count re-mained below that of the controls. In effect, within total T lympho-cytes, the number of T CD8�, but not T CD4�, B lymphocytes, or NKcells prevailed over basal values (Fig. 1 and Table 2), and withinCD8� T lymphocytes, CD8�KIR2D� T cells increased from days 0 to365.

Furthermore, KIR2D� cells are apparently not linked to themost prevalent liver transplantation indications (cirrhosis andHCV preinfections) or to variations in immune suppression. Ef-fectively, in these pathologies KIR2D� cell profiles resemblingthat of the total cohort of patients, and mainly those ofCD8�KIR2D� T lymphocytes in HCV preinfected patients, agreewith the undetectable reported induction of KIR on HIV or CMV-specific CD8� T cells in humans or mice affected by bacterial orviral infections [47]. Likewise, a dependence on immunosup-pressive therapy can be ruled out because overall patients re-

ceived analogous prophylactic initial doses of immunosuppres-

sive drugs or antirejection therapy. Importantly, these treatmentsdid not lead to remarkable variations in NK cell recuperation inpatients suffering or not suffering fromearly AR; the decreased rateof CD8�KIR2D� T cells in patients experiencing early AR was evi-ent before the appearance of these episodes, and both KIR2D cellatterns evolved similarly in patients who received cyclosporin oracrolimus.

Although no consensus exists regarding whether single C-igands influence the short- or long-term liver transplant out-ome [4–6,39], our data seem to indicate that the HLA-C geno-ype of the recipient might at least condition the emergence ofIR2D� NK cells following liver transplantation. The main recov-ry of KIR2D� NK cells in C2/C2 patients who suffer from acute

rejection is in line with the graft failure observed in C2/C2patients undergoing liver transplantation or HSCT [5,30] andwith the previous hypotheses regarding the C-ligand impact onNK cell repertoire shaping [29,40,42,43,48].

In summary, for the first time, the systematic screening ofIR2D� cells performed in this study is consistent in showing aiasing of the KIR2D� NK and CD8� T cells recovering in liver

transplants with or without AR as evidenced by the late emer-gence of NK cells in C2/C2 patients suffering from early AR andthe early increase of CD8�KIR2D� T-cell proportions in patientshowing increased transplant success. Therefore, although fur-her studies are needed to confirm these results in other series,he monitoring of KIR2D� cells, together with HLA-C genotypingof patients, might be helpful in liver transplant follow-up anddetermining which patients could have a greater short-termlikelihood of accepting a liver allograft. New experiments areunderway to evaluate the implication of KIR2D� cells in theallogenic response, with particular attention to their liver infil-tration and functional activity.

Acknowledgments

Thisworkwas supported by theCIBERehdprogramand07/1224and 10/01964 projects from the Fondo de InvestigaciÔn Sanitaria,Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo,Spain, and Caja Murcia (2008–2010) aid, and by FundaciÔn SÊneca,Murcia, Spain (Project 04087/GERM/06). The research work ofMarÎa RocÎo LÔpez-Alvarezwas financed by the CIBERehd Program.The authors also thank the following individuals for their kindcollaboration: Isabel MarÎn, Beatriz Las Heras, and Sandra Sorianofor technical assistance; Carmen MÂrquez for viral infection diag-nosis; the Immunology, Digestive, Surgery, and Microbiology Ser-vices of the University Hospitals Virgen de la Arrixaca in Murcia;and Gregorio MaraÒÔn in Madrid.

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