doi:10.1182/blood-2006-08-040238Prepublished online February 1, 2007;   

 Truong, Ioannis Theodorou, Francoise Barre-Sinoussi, Gianfranco Pancino and Pascale PaulSophie Ravet, Daniel Scott-Algara, Elodie Bonnet, Hung Khiem Tran, Ton Tran, Ngai Nguyen, Lien Xuan cell activation in HIV-exposed uninfected individualsDistinctive NK cell receptor repertoires sustain high-level constitutive NK

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Distinctive NK cell receptor repertoires sustain high-level constitutive NK cell activation

in HIV-exposed uninfected individuals

Short title: NK cells in HIV-exposed uninfected individuals

Sophie Ravet1, 2, Daniel Scott-Algara3, Elodie Bonnet1, Hung Khiem Tran4, Ton Tran4, 6,

Ngai Nguyen5, Lien Xuan Truong4, Ioannis Theodorou6, Françoise Barré-Sinoussi3,

Gianfranco Pancino3, Pascale Paul1, 2*

1- Laboratoire Exploration NK, Laboratoire d’Hématologie, CHU Conception,

Assistance Publique Hôpitaux de Marseille, 147 boulevard Baille, 13005 Marseille,

France

2- UMR-S 608 INSERM, URF de Pharmacie Université de la Méditerranée, 27 Bd Jean

Moulin, 13385 Marseille 5, 13005 Marseille

3- Unité de Régulation des Infections Rétrovirales, Institut Pasteur, Paris France

4- Pasteur Institute, Ho Chi Minh City, Vietnam

5- Binh Trieu Hospital, Ho Chi Minh City, Vietnam

6- INSERM U543, CHU Pitié-Salpêtrière, Paris, France

Corresponding author Pascale Paul Laboratoire Exploration NK, Hematology unit Hôpital de la Conception 147 Bd Baille 13005 Marseille, France Tel. 33 (0) 49 13 83 920 Fax 33(0) 49 13 83 012 Email: pascale.paul@ap-hm.fr

Word counts: Abstract : 199 Text: 4877 Scientific heading: Immunobiology

Blood First Edition Paper, prepublished online February 1, 2007; DOI 10.1182/blood-2006-08-040238

Copyright © 2007 American Society of Hematology

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Abstract

We have previously associated high natural killer (NK) cell activity and protection

against HIV-1 infection in Vietnamese exposed uninfected intravenous drug users (EUs).

Considering that activating and inhibitory signals sensed by NK cell receptors regulate NK

cell activation, we performed phenotypic and qRT-PCR transcript analyses of the NK cell

receptor (NKR) repertoire in 25 EUs, 19 HIV+ intravenous drug users and 26 uninfected

blood donors. Although NK cell activation was not linked to a unique NKR repertoire in EUs,

various patterns consistent with NK cell activation were detected in EUs: high

KIR3DS1/KIR3DL1 ratio associated with down-regulated KIR3DL1 transcript levels,

KIR2DL3+ low affinity receptor expansion associated to group HLA-C1 ligand in 2DS2-

/2DL2- EUs, enhanced NKG2C/NKG2A ratio, and increased CD69 expression. Remarkably,

EUs exhibited high constitutive degranulation activity in the absence of exogenous

stimulation, as shown by the CD107a assay. Furthermore, CD161 expression was increased

within the CD107a+ NK cell compartment. Our results suggest that in response to viral

exposition, particular genetic and/or regulated features of the NKR repertoire of EUs

contribute to their high constitutive NK cell potential. This might allow NK cells to generate a

more rapid and effective immune response to HIV-1, thereby contributing to prevention

toward infection.

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Introduction

Natural killer (NK) cells are key actors in innate host immune responses to a variety of

pathological challenges. NK cell activation results from integration of signals induced by the

interaction of a highly diversified repertoire of inhibitory and activating NK cell receptors

(NKRs) with constitutive or inducible ligands expressed on target cells, including virus-

infected cells1-5. Inhibitory receptors essentially recognize specific subsets of classical and

non-classical HLA class molecules, and include KIR-L, ILT2/CD85j, and heterodimeric

CD94/NKG2A NKRs. Inhibitory KIR2DL1 recognizes HLA-C group 2 alleles (HLA-C2:

Asn77Lys80), while KIR2DL2 and KIR2DL3 both recognize HLA-C group 1 alleles (HLA-

C1: Ser77Asn80) but with different affinity2,6. KIR3DL1 recognizes the HLA-Bw4 group of

alleles 7and KIR3DL2 HLA-A11/HLA-A3. Recognition of the relevant HLA class I molecule

by KIR-L receptors delivers NK inhibitory signals, whereas non recognition allow

stimulatory signals to activate NK cells. MHC-KIR interactions shape the NK cell repertoire

and calibrate NK cell potential to control infectious, tumoral or alloreactive challenges5,8,9 . In

contrast to KIR-L, activating KIR-S counterparts can deliver activating signals to NK cells,

but their physiological ligands remain mostly uncharacterized. Recent evidence suggest that

KIR2DS1 and KIR2DS2 respectively share the capacity to bind HLA-C2 or -C1 ligands with

their inhibitory counterparts in vitro, but with lower affinity10. Receptors delivering activating

signaling include NK-specific natural cytotoxicity receptors (NCRs), in addition to receptors

more broadly distributed among T lymphocyte subsets, such as activating KIR-S,

CD94/NKG2C, NKG2D, CD226/DNAM and CD161/NKRP1. Activated NK cells mediate

various effector mechanisms, including antibody-dependent and natural cytotoxicity, and

secretion of chemokines and cytokines with antiviral activity1,2,4,5. In addition to these effector

functions, crosstalk between NK cells and antigen-presenting cells, and cytokine secretion by

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NK cells, influence the efficiency of adaptive immune responses, thereby constituting a major

link between innate and adaptive immunity6,7.

NK cells therefore have the potential to contribute to the prevention and control of

viral infections, including HIV-1 infection. The NK cell phenotype and functions are altered

in HIV+ patients7-14. Up-regulation of inhibitory receptors in viremic patients has been

proposed as a basis for the inhibition of NK cell activity observed in such subjects12,15,16.

Decreased expression of NKp30 and NKp46 NCR activating receptors has also been linked to

impaired cytolytic potential in viremic patients relative to HAART-treated aviremic subjects

and to healthy individuals11-13. Partial quantitative and functional reconstitution of NK cells

can be obtained during HAART, thus linking NK cell suppression to HIV replication14-17. A

role of NK cells in controlling HIV disease progression is suggested by the association

between delayed progression to AIDS and concomitant presence in the host genome of genes

that contribute to the HLA-Bw4 allelic group and activating KIR3DS1 NK receptor genes18.

The rare individuals who are naturally resistant to HIV-1 infection represent a unique model

in which to examine the role of innate immunity in the control of HIV-1, with potential

implications for preventive and therapeutic strategies. We have previously observed enhanced

NK cell cytotoxic activity and cytokine and β-chemokine production in Vietnamese

intravascular drug users who remained uninfected despite several years of high-risk exposure

to HIV-1, when compared with HIV+ drug users (IDU) and low-risk uninfected blood

donors19. In contrast, NK cell activity was low in some drug users who seroconverted during

the study19. These results suggested that NK cells contribute to the protection of these

exposed uninfected individuals (EUs). A recent study analyzing genomic HLA-KIR

combinations in African female sex workers suggests that resistance to HIV-1 infection

associates with inhibitory KIR in absence of their HLA ligand20. The present study was

designed to investigate whether specific patterns of NK cell receptor expression associate

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with higher NK cytotoxic and secretory activities in Vietnamese EU IDUs. We therefore

conducted comparative genomic, transcriptional and phenotypic analysis of the NK cell

repertoire, in association with the functional assay of the CD107a degranulation potential of

NK cells, in EUs, HIV+ IDUs, and uninfected low-risk blood donors.

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Materials and Methods

Study population

Twenty-five exposed uninfected (EUs) intravascular drug users (IDU) from a previously

described cohort recruited in the Binh Trieu Hospital in Ho Chi Minh City19,21 were included

in this study. Risk factors for HIV exposure, the high seroprevalence of other blood-borne

viruses such HBV, HCV and HTLV, and the follow-up for this sub-study were previously

described19. ELISA confirmed the HIV-1 negative status of EUs and PCR as described21.

Nineteen HIV+ intravascular drug users were studied (HIV+ IDUs) and offered free diagnosis,

medical follow-up and treatment for opportunistic infections. Twenty-six low-risk uninfected

controls (C) were recruited from the Blood Bank of Ho Chi Minh City. Informed consent was

obtained from each participant at recruitment. All the subjects were males, and there was no

age difference among the study groups. The Ethics Committee of Binh Trieu Hospital, Ho

Chi Minh City, approved the protocol. Peripheral blood mononuclear cells (PBMC) were

recovered from blood samples after Ficoll-Hypaque gradient and cryopreserved in 90% FCS,

10% DMSO. When PBMC or NK cell counts were a limiting factor, in particular in HIV+

individuals, functional, phenotypic and transcriptional analysis of NK cells were privileged

over DNA extraction as a source of informative data.

HLA and KIR genotyping

HLA genotyping was performed with a reverse dot blot assay using a commercial kit and

according the manufacturer's instructions (Innogenetics Belgium). Due to the limited

availability of biological samples, KIR genotyping could only be performed on 14 EUs, 8

HIV+ IDUs, and 12 controls. A reverse hybridization assay read using Luminex technology

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was used for KIR genotyping according to manufacturer’s instructions (One Lambda, Inc.,

CA USA).

RNA purification and real time RT-PCR analysis of KIR transcript levels

Total RNA was isolated from PBMC using RNeasy mini kits (QIAGEN Inc., Valencia, CA,

USA) including a DNase I digestion step removing genomic DNA. Total RNA (5µg) was

converted to cDNA using 200 U of Supercript II reverse transcriptase (Invitrogen). Real-time

PCR amplification was performed with the FastStart DNA MasterPLUS SYBR Green I kit as

recommended by the manufacturer, on a LightCycler instrument (Roche). Cycling conditions

were 10min at 95°C (hot-start PCR), followed by 40 cycles, 10s at 95°C (denaturation), 10s at

62°C (annealing) and 20s at 72°C (elongation). Melting curve analysis was performed to

check the specificity of amplification. Reported values are relative numbers of specific

transcripts detected per 106 GAPDH transcripts. KIR expression was analyzed with primers

specifically designed to discriminate KIR locus, as follows (forward and reverse sequences):

KIR2DL3 5’-CTCCTTCATCGCTGGTGCTG-3’ and 5’-

GGCAGGAGACAACTTTGGATCA-3’; KIR2DL4 5’-CTCCCAGAGCTCCTTTGACATC-

3’ and 5’-GAGCCGAAGCATCTGTAGGTCT-3’; KIR3DS1 5’-

CCAAGCTCCAAATCTGGTAACCT-3’ and 5’-TCAGAATCCTCGCTGTTCACTTCT-3’;

KIR3DL1 5’-GGACATCGTGGTCACAGGTCC-3’ and 5’-

CACTGAGGTCCCAATCAGAATG-3’; KIR3DL2 5’-CCAACTTCTCCATCGGTCCCT-3’

and 5’-GTTGACCTTGGGCACTGCAC-3’; GAPDH 5’-

GGTGGTCTCCTCTGACTTCAACA-3’ and 5’-GTTGCTGTAGCCAAATTCGTTGT-3’.

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Phenotypic analysis

The percentage of CD3-CD56+CD16+ NK cells among total lymphocytes and the size of each

NKR+ NK cell subset were determined by three-color flow cytometry (Beckman-Coulter).

Eleven distinct NK cell subsets were defined as NK cells (CD3-CD56+) expressing specific

NKRs. The following antibodies were used: FITC-conjugated anti-CD3 (IgG1, UCHT1);

PC5-conjugated anti-CD56 (IgG1, N901); PE-conjugated: anti-CD158a/h (clone EB6, IgG1

recognizing KIR2DS1/2DL1), anti-CD158b1/b2/j (clone GL183, IgG1 recognizing

KIR2DS2/2DL2/2DL3), anti-CD158e1/e2 (clone Z27.3.7, IgG1, recognizing

KIR3DL1/KIR3DS1), anti-CD158i (clone FES172, IgG2a, recognizing KIR2DS4), anti-

CD25 (IgG2a, B.1.49.9), anti-CD161 (IgG1, 191B8), anti-NKp30 (IgG1, Z25), anti-NKp46

(IgG1, BAB281), anti-CD85j (IgG1, HP-F1), anti-CD159a (IgG2b, Z199), (all from Beckman

Coulter), anti-CD69 (IgG1, FN50, BD-Pharmingen), and anti-NKG2C (IgG1, 134591, R&D

Systems).

Flow cytometer analysis of NK function using the degranulation marker

CD107a/LAMP-1 (lysosome-associated membrane protein 1)

CD107a expression was used to measure NK cell degranulation, as described22,23. PBMC

were incubated with or without HLA class I -negative K562 target cells (ATCC) at an E: T

ratio of 2:1 for 3 hours. CD107a-PC5 antibody (IgG1, H4A3) and GolgiStop reagent diluted

at 1:150 (BD/Pharmingen) were added directly to the cultures. Cells were then stained with

anti-CD3-FITC (IgG1, UCHT1), CD56-PC7 (IgG1, N901) prior to FACS analysis. Additional

NKR-PE staining was performed with anti-CD85j, anti-CD159a, anti-CD161 (Beckman-

Coulter), anti-NKG2C (R&D System), and anti-CD69 (BD Pharmingen) antibodies. All

samples were analyzed by means of four-color flow cytometry and CXP software (Beckman-

Coulter).

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Statistical analysis

Analyses were performed with Prism software (GraphPad 4.0b) implementing the

nonparametric Kruskall-Wallis test followed by Dunn’s post-test to compare three or more

continuous variables, the Mann-Whitney test to compare two unpaired groups, and the

Wilcoxon matched pairs test for the CD107a degranulation assay results.

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Results

Distinctive features of the KIR repertoire in exposed uninfected intravascular drug

users

The size of the NK cell compartment did not differ between EUs and controls (median 16%

and 16.4% respectively), whereas NK cell percentages were lower in HIV+ IDUs (HIV)

(median 11.6%, p<0.05, Figure 1A).

KIR+ NK cell subsets distribution was analyzed by flow cytometry in 25 EUs (Table 1), 19

HIV+ and 26 unexposed Vietnamese control donors. Four KIR+CD3-CD56+ NK cell subsets

were evaluated using antibodies EB6 (CD158a/h or KIR2DL1/2DS1), GL183 (CD158b1/b2/j

or KIR2DL2/2DL3/2DS2), Z27.3.7 (CD158e1/e2 or KIR3DL1/3DS1) and FES172 (CD158i

or KIR2DS4). As previously described in Caucasians24, high inter-individual heterogeneity

was observed in the distribution of each KIR+ NK cell subsets. Still, no significant differences

in the size of these KIR+ NK cell phenotype subsets were observed among the three

Vietnamese groups (data not shown).

As phenotypic analysis of the KIR repertoire with currently available mAbs cannot

discriminate between inhibitory and activating KIR-expressing NK cell subsets, we further

performed real-time PCR analysis of the KIR repertoire to quantify the mRNA transcripts

corresponding to each of the five activating KIR-S and six inhibitory KIR-L receptors. In

addition, HLA and KIR genotyping was also performed in 14 EUs, 8 HIV+ IDUs and 12

controls for which enough cells were available for DNA analysis (Table 1).

Overall, the KIR gene frequencies observed in our control group (Figure 1B) were

comparable to those previously reported in Vietnamese blood donors25. Moreover, we show

that KIR transcripts frequencies were mostly similar in EUs and controls (Figure 1B). In

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contrast, transcript frequencies of given KIRs were selectively altered within the HIV+ group

(Figure 1B).

Remarkably, KIR2DL4 and KIR3DL2 transcripts, corresponding to core KIR genes found at

the genomic level in all individuals, were found in all EUs and controls, but were

undetectable in some HIV+ individuals (Figure 1B). Similarly, the core KIR3DL3 gene was

detected in 100% of controls but the corresponding transcripts were undetectable in 22% of

HIV+, 40% of EU and 29% of controls tested. The dissociation between KIR3DL3 gene

content and transcript frequency (p<0.05) was thus not specific of a given group (Figure 1B).

Transcript frequencies of other KIRs showed significant differences in HIV+ subjects in

comparison with EUs and controls (Figure 1B). First, the frequency of individuals with

detectable KIR3DL1 and KIR2DL3 transcripts were lowered in HIV+ IDUs (p=0.0007 and

p=0.0174 respectively). Similarly, frequency of individuals expressing KIR3DS1 transcripts

tended to be lower in HIV+ individuals (p=0.0992). Our transcriptional analysis included

design of specific primers that discriminate transcripts corresponding either to the KIR1D null

allele that is not expressed as an NK receptor or to the group of KIR2DS4 alleles that

contribute to cell surface expression of the KIR2DS4/CD158i receptor26. Using this

approach we showed that frequency of KIR2DS4 transcripts coding for KIR2DS4

receptor was higher in HIV+ IDUs than in EUs and controls (p=0.035, Figure 1B).

When allowed by cell or DNA availability, we further investigated whether the absence of

detectable KIR3DL1 or KIR3DS1 transcripts was associated to the absence of the gene or

whether it may correspond to lack of transcription in individuals who bear the gene. For

KIR3DS1, a good correlation between gene content and transcript detection was observed in

all groups. In particular, HIV+ IDUs in whom KIR3DS1 transcripts were undetectable were

also typed as KIR3DS1 negative at the gene level. In contrast, while the presence of the

KIR3DL1 gene was always associated to KIR3DL1 transcripts in controls, 3 out of 8 HIV+

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IDUs and 3 out of 14 EUs genotyped were shown to be KIR3DL1-positive at the gene level

but lack the corresponding transcripts.

We further analyzed quantitative differences in the levels of KIR transcript expression in the

three groups (Figure 1C and results not shown). KIR2DL4 and KIR3DL2 transcripts levels

were lower in HIV+ subjects than in controls or EUs (p<0.05, Figure 1C). On the other hand,

KIR3DL3 transcript levels were also lower in EUs than in HIV+ and controls (p<0.05, data not

shown).

In contrast, in HIV+ subjects. KIR2DL3 transcript levels were lowered in reference to EUs and

controls (p<0.05 and p<0.001 respectively). Combined KIR2DS2-/2DL2-/2DL3+ transcripts

pattern tended to be more frequent in EUs than in HIV+ or controls (p=0.10, Fig 1B). We

further observed higher KIR2DL3 transcript levels in these KIR2DS2-/2DL2-/2DL3+ EU

individuals (p<0.05, Figure 1C). Notably, 5 EUs exhibiting more than 50% GL183+ stained

NK cells were further identified as KIR2DL3+and 2DL2-/2DS2-, both at the gene and

transcript levels (Figure 1C). KIR2DL3 was thus the only KIR gene associated to the GL183+

phenotype in these individuals.

Further analysis of HLA groups associated with the KIR repertoire in EUs showed that

groups HLA-C1/C2 and HLA-C1/C1 were equally represented, and that only 2 EUs were

homozygous HLA-C2/C2 (Table 1). The highly represented GL183+ NK cell subsets in

KIR2DL3+/2DL2-/2DS2- EUs was always associated with presence of the HLA-C1 ligand in

these individuals.

KIR3DL1 transcript levels also tended to be lower in EUs than in HIV+ subjects and controls

(Figure 1C), although statistic significance was not reached. Accordingly, and given the high

KIR3DS1 transcript levels in some EUs, the ratio between activating KIR3DS1 and inhibitory

KIR3DL1 transcript levels was higher in some EUs, and particularly elevated at values never

observed in tested controls in three EUs (Figure 1C; n° 6, 7, 16 in Table 1).

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Increased expression of NK cell activation markers in EUs

The NK cell repertoire was further characterized by phenotypic analysis of NK cell subsets

expressing activating and inhibitory NKRs, CD25 and CD69 activation markers (Figure 2A,

B and data not shown). NKp30 expression was found on all NK cells and at comparable

levels in EUs and controls. The level of NKp30, assayed by mean fluorescence intensity of

NKp30 staining (mfi), was lower in HIV+ than in EUs and controls (p<0.05, Figure 2A). In

contrast, no significant difference in NKp46 (Figure 2A) nor NKG2D and DNAM expression

(data not shown) was observed among the three groups. Although the size of the CD161+ NK

cell subset was not different among the three groups, CD161 mean fluorescence in

CD161+CD3-CD56+ cells was significantly higher in EUs than in controls (p<0.05, Figure

2A). While the NK-cell surface expression of the CD25 activation marker was comparable in

all tested groups (data not shown), higher percentage of CD69+ NK cells was observed in EUs

and HIV+ as compared to controls (p<0.05, Figure 2A). Overall, CD85j, NKG2A and

NKG2C expression on NK cells did not differ significantly among the groups. Nevertheless,

expression of the activating NKG2C subunit of the CD94/NKG2 receptor was inversely

correlated to that of its inhibitory NKG2A counterpart in EUs, but not in the other two groups

(p<0.05, Figure 2A).

Strong constitutive activation of NK cells in EUs.

We then investigated the granule exocytosis pathway characterizing NK cell activation by

evaluating CD107a+ expression within CD3-CD56+ NK cells23, both without stimulation and

in the presence of stimulatory K562 cells (Figure 3).

Without stimulation, the size of the activated CD107a+ NK cell subset was much larger in

EUs (median: 34%, 25-75% interquartile ranges: 31-50%) than in HIV+ (10%, 5-19%,

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p<0.01) and controls (8%, 1.7-12%, p<0.001) (Figure 3A, C). Kinetic analysis of CD107a

degranulation at 10 min, 1h, 2h and 3h showed that degranulation of unstimulated EU NK

cells occurred between 10 min and 1 hour, in contrast to 2 hours in cells from controls (data

not shown). Median percentage of CD107a+ cells among control CD3-CD56+ NK cells was

increased by K562 stimulation (from 8% to 17%, p<0.05), but remained unchanged in HIV+

individuals (Figure 3A).

Upon stimulation by K562 cells, median percentage of CD107a+ NK cells remain

significantly higher in EUs (39%, 24-46%) than in HIV+ (9.5%, 8-15%, p<0.001). and

stimulated controls (17%, 9-25%, p<0.01, Figure 3A).

We further analysed variations in intensity of CD107a expression, as assayed by mfi of

CD107 staining within the activated CD3-CD56+ NK cell subset. Stimulation with K562 cells

induced increased CD107a mean fluorescence staining intensity within CD107+ NK cells, in

controls as well as in EUs (Figure 3B). In contrast, the mfi of CD107a+ NK cells from HIV+

individuals was unchanged after K562 stimulation (Figure 3B).

Increased CD161 expression is associated with the CD107a+ activated NK compartment

Finally, we examined the NK cell surface expression of CD69, CD85j, NKG2A, NKG2C and

CD161 within the activated CD107+CD3-CD56+ NK cell compartment, both in the presence

and absence of stimulatory K562 target cells. Overall, the distribution of CD85j, NKG2A, and

NKG2C expression among CD107a+CD3-CD56+ NK cells did not differ significantly among

the three study groups (data not shown). Median percentages of CD69 expression among

CD107a+ NK cells tended to be higher in EUs (40%) than in HIV+ (20%) and controls (28%)

in the absence of K562 stimulation, and were induced to similar levels in the three groups

after K562 stimulation (data not shown).

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In contrast, CD161 expression differed significantly between EUs and the other groups

(Figure 4). In the absence of K562 stimulation, the percentage of CD161+ cells among

CD107+CD3-CD56+ NK cells were higher in EUs than in controls (p<0.05) and HIV+ subjects

(p<0.01, Figure 4A, B). In addition, the intensity of CD161 staining in unstimulated

CD161+CD107+CD3-CD56+ NK cells (mfi) was comparable in EUs and controls and

significantly lowered in HIV+ (p<0.05, Figure 4B, C), while not affected by K562 stimulation

in any of the three groups (data not shown). Among CD161+ NK cells, intensity of CD161

staining (mfi) in EUs tended to be higher in the CD107a+ NK subset than in the CD107a- NK

subset (p=0.12, Figure 4D). In contrast, CD161 expression levels were similar in the CD107a+

and CD107- NK cell compartments in HIV+ and controls subjects (Figure 4D).

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Discussion

The role of NK cells in controlling HIV infection has mainly been investigated in infected

individuals. Thus, while considerable information has been gained on NK cell dysfunctions

associated with HIV infection and viremia, the role of NK cells in protection against HIV

infection remains to be clarified. Natural resistance to infection among exposed uninfected

individuals may be due partly to efficient early innate antiviral responses. Indeed, we

previously showed that highly exposed uninfected Vietnamese IDUs have higher NK

cytokine/chemokine secretion capacities and lytic activity19. More recently, Montoya et al

reported increased interferon-γ (IFN-γ) production by NK cells from uninfected individuals

sexually exposed to HIV-127.

The present study was designed to further characterize the NK effector potential of

Vietnamese EU IDUs using a recently described degranulation functional assay22 and to

investigate whether increased levels of activation could be associated with peculiar features of

the NK cell receptor repertoire in EUs. In addition to the phenotypic analysis of KIR+ NK cell

subsets, our study is to our knowledge the first to provide a quantitative analysis

discriminating KIR transcripts in virally exposed IDU individuals in reference to low risk

control donors.

Using the CD107a NK functional assay22, we found that EU NK cells exhibit a high

constitutive degranulation potential. Indeed, degranulation occurred rapidly in vitro, without

the need for exogenous target cell stimulation. This high constitutive activity, which likely

reflects a strong in vivo activation status of the NK cell compartment in EUs, was not

observed in HIV+ or in low risk controls recruited in the same environment. In other studies,

high levels of CD107a expression on NK cells from HIV viremic subjects was associated

with a decreased number of CD3-CD56+CD161+ NK cells28. Up-regulation of CD107a has

been shown to correlate with NK cytokine secretion and cytolytic activity22,23,28. This is in

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line with our previous observation of that EU exhibit a higher NK cytolytic potential cells

toward Daudi and K562 targets and an enhanced NK-mediated production of IFN-γ, TNF-α

and CCL3-519. The additional functional read out of NK cells provided in this study, therefore

stresses the fact that EU’s NK cells have a particularly high constitutive potential to display

immediate effector functions upon infectious challenge, that possibly contribute to the

protection of EUs against HIV infection.

The phenotypic analysis of the NK cell repertoire identified particular NK cell receptor

patterns that may sustain constitutive NK cell activation in EUs, such as enhanced CD69 and

CD161 expression. In particular, intensity of CD161 staining on NK cells was higher in EUs

than in HIV+ individuals and controls. Furthermore, following stimulation, CD161 was

preferentially expressed by the activated CD107a+ NK cell subset. Number of CD161+ NK

cells were shown to be reduced in HIV infected viremic subjects29, but no correlation with

NK functional defects observed in HIV+ patients has yet been established. CD161

engagement appears to have a complex role in the modulation of immune functions. Both

activating and inhibitory effects have been described in mice after CD161 receptor

engagement30. In humans, LLT1, an inhibitory ligand of human CD161, was shown to

modulate NK cell activity31,32. CD161 expression was also involved in the triggering of NK

cell cytotoxicity towards the Daudi tumor cell line33. Interestingly, we have previously shown

that EU NK cells display substantial cytotoxic activity against Daudi cells19. Although direct

evidence implicating CD161 in enhanced NK cell activation is still lacking, these

observations suggest that CD161 expression may contribute to the NK cell activation status,

at least in HIV-exposed uninfected subjects.

Although down-regulation of NKp30 was confirmed in HIV+ individuals12, no major

alteration of NCRs was found in EUs in comparison to low-risk controls, thus suggesting that

NK activation in EUs was not associated with increased expression of activating NKp30 or

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NKp46 receptors. Other features of the NK cell repertoire that senses virally induced

modification of HLA class I expression may sustain EU’s NK cell activation. Cell activity

against HIV-1-infected blasts was shown to be partly dependent on NK cell recognition of

HIV-modulated HLA-E expression34. This recognition is mediated by the heterodimeric

receptor CD94, in association with either its inhibitory NKG2A subunit or its activating

NKG2C subunit. In some EUs the stimulatory NKG2C to inhibitory NKG2A ratio was

inverted, in favour of the activating subunit. While no significant change in the relative size of

the CD94+ NK subset was found in EUs, this suggests that HLA-E interaction with NK cells

expressing the CD94/NKG2C activating receptor may be dominant in some EUs. Such a

switch from inhibitory to activating NKG2 subunit expression of the CD94/NKG2 receptor

for HLA-E has been linked to concomitant infections in HIV+ individuals and may be driven

in EUs by exposure to viral infections other than HIV35.

Although the phenotypic distribution of NK cells expressing KIR and CD85j MHC-class I

receptors was not profoundly affected in EU, further discrimination of the activating and

inhibitory KIR genes that contribute to the phenotype of KIR+ NK cell subsets, by locus-

specific real-time PCR transcript quantification, revealed qualitative and/or quantitative

differences between EUs and the other two groups of subjects. In contrast to the other two

study groups, high percentages of GL183-positive NK cells in EUs correlate with the

expansion of KIR2DL3 transcripts in the absence of KIR2DS2 and KIR2DL2 transcripts. In

addition, this expansion of KIR2DL3 transcripts was observed in EUs displaying either HLA-

C1/C1 or C1/C2 allelic specificities. As the inhibitory KIR2DL3 receptor binds to its ligand

HLA-C1 with lower affinity than KIR2DL2, KIR2DL3 expression in the absence of

KIR2DL2 expression may favour NK cell activation in EUs, by a mechanism similar to that

proposed for KIR2DL3-HLA-C1 in the resolution of HCV infection36. Indeed, NK cell

interactions involving KIR2DL3-HLA-C1 have been shown to directly influence the

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resolution of hepatitis C virus (HCV) infection in Caucasians and African Americans36. This

led to the concept that not only activation but also lowered NK inhibitory responses may be

an advantage in the control of viral infection. KIR2DL2/2DL3 heterozygosity in absence of

HLA-C1 ligand has been recently reported to be associated to the seronegative status of

exposed African sex workers20. In our study, all KIR2DL2/2DL3 heterozygous EUs were

homozygous HLA-C1/C1 or heterozygous HLA-C1/C2. Moreover, the two C2/C2 EUs were

typed as homozygous for KIR2DL3. Therefore, although our sample is clearly too limited to

establish any firm statistics on HLA-KIR combinations in EUs, the KIR2DL2/2DL3

heterozygosity in absence of HLA-C1 does not appear to contribute to resistance in

Vietnamese EU IDUs.

Resistance to HIV-1 infection among African sex workers was also associated with KIR3DL1

homozygosity in the absence of its cognate HLA-Bw4 ligand20. No such trend was identified

in Vietnamese EUs, as homozygous HLA-Bw6 and Bw4/Bw6 were equally represented in

KIR3DL1 homozygous EUs (Table 1). Interestingly, in some EUs, concomitant expression of

lowered inhibitory KIR3DL1 transcript levels and high activating KIR3DS1 levels resulted in

a high KIR3DS1/KIR3DL1 ratio that may confer an enhanced activating NK cell repertoire

profile to these EUs. Indeed, very recent data suggest that KIR3DS1 associates with DAP12

and triggers both cytolysis and IFN-γ production37. Previous genetic studies have linked

KIR3DS1 and HLA-Bw4 expression to slow progression toward AIDS18,38. However, in our

study, strongest KIR3DS1 expansion in EUs occurred in HLA-Bw6 individuals rather than in

homozygous HLA-Bw4 EUs who were not found in our study population (Table 1).

Furthermore, it was recently suggested that activating KIR3DS1 receptor does not recognize

HLA-Bw437,39.

Although the number of individuals analyzed was too small to draw statistically powerful

conclusions, combined transcriptional and genomic analysis suggested that KIR3DL1 and

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core KIR2DL4 and KIR3DL2 transcription might be specifically abolished in some HIV-

exposed individuals. Indeed, KIR3DL1 transcripts reflected the KIR3DL1 gene content in

controls, whereas the absence of KIR3DL1 transcripts was observed in presence of the

KIR3DL1 gene in some HIV+ and EU IDUs. Although the possibility that KIR3DL1 gene

polymorphisms specific to HIV-exposed Vietnamese were not detected by our RT-PCR is not

excluded, since PCR primers designed to specifically detect KIR3DL1 transcripts did not

allow their use for gene amplification, this is unlikely considering that KIR3DL1 genotype

was confirmed in these individuals by commercial available kits. Our transcriptional approach

of the KIR repertoire, although limited by the number of individuals studied, thus suggests

that genomic KIR content is not always associated to transcript detection in HIV-exposed

populations, and that mechanisms regulating KIR transcripts may occur in exposed

individuals. Representation of KIR transcripts might not only represent a down- or up-

regulation but a selection at infection. Interestingly, the frequency of individuals that exhibit

KIR2DS4 transcripts encoding the KIR2DS4 receptor was significantly increased in HIV+

IDUs, suggesting that the KIR2DS4 bearing haplotype, often associated to lack of other

activating KIR-S gene content8, might be more prevalent in HIV+ individuals. A challenging

issue to be addressed in wider population studies would be to investigate whether KIR

regulation is selected at infection and to what extent regulatory mechanisms can be

specifically related to HIV or to concomitant co-infections occurring in exposed individuals.

In conclusion, our results and data from other studies of HIV+ patients suggest that the NK

cell receptor repertoires in EUs may be quite different from those of HIV-1-infected

individuals, whether they are viremic or non progressors13,18,28,29,40. Several mechanisms,

including cellular restriction of HIV replication, T cell activity and specific IgA responses

have been proposed to explain resistance to HIV infection41, but it is more likely that complex

genetic and/or selected mechanisms operate independently or in synergy to confer individual

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resistance to HIV-1. HIV-exposed uninfected individuals are a heterogeneous population, and

it is therefore not surprising that the high level of NK cell activation that characterizes the

Vietnamese EUs studied here was associated with diverse NK receptor repertoires. Yet, in

some EUs, no particular pattern of NK receptors could be identified. The constitutive highly

activated status of NK cells from EUs may also be dependent on the HLA-NK receptor

genetic background and/or on selective induction of NK receptors that endow NK cells with

the capacity to mount an effective defence against HIV-1. The increased NK cell activity that

we found in Vietnamese EUs may reflect more general immune activation resulting from

several factors including exposure to multiple viruses42. However, HIV-1-infected IDUs

exposed to similar environmental conditions and to multiple viral infections19,43 have different

NK cell receptor patterns. Therefore, our data strongly suggest that the increased NK cell

activity in virally exposed EUs is linked to particular genetic backgrounds that adapt the

regulation of diverse NK cell receptors upon viral challenge, thereby shaping a constitutively

highly activated NK cell status that may contribute to protection of EUs toward HIV-1

infection.

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Acknowledgments

This work was supported by grants from Agence Nationale de Recherche sur le Sida,

Assistance publique Hôpitaux de Marseille (AP-HM), INSERM (action thématique concertée

ATC Biothérapies), Ministère de la Recherche Réseau Genhomme. Authors' contributions:

PP, FB-S, DS-A and GP conceived the study. PP, DS-A and GP contributed to the

experimental design and coordination of the study. LXT and NN performed the recruitment of

the study subjects and coordinated the study in Vietnam. SR, EB and HKT performed the

transcriptional, phenotypic and functional analyses. IT and TT contributed the HLA and KIR

genotyping. PP, SR, GP and DS-A wrote the manuscript, IT to its critical correction. All

authors read and approved the final manuscript. We thank all the physicians, family

practitioners and medical staff from the Binh Trieu Hospital and the Pasteur Institute of Hô

Chi Minh City who took part in this study. We thank Denis Reviron, Christophe Picard and

Coralie Frassati for their helpful advice in KIR genotyping and members of the Hematology

unit and AP-HM for support and availability of flow cytometer facilities and Françoise

Dignat-George for providing helpful comments on the manuscript.

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20. Jennes W, Verheyden S, Demanet C, et al. Cutting Edge: Resistance to HIV-1 Infection among African Female Sex Workers Is Associated with Inhibitory KIR in the Absence of Their HLA Ligands. J Immunol. 2006;177:6588-6592. 21. Truong LX, Luong TT, Scott-Algara D, et al. CD4 cell and CD8 cell-mediated resistance to HIV-1 infection in exposed uninfected intravascular drug users in Vietnam. Aids. 2003;17:1425-1434. 22. Alter G, Malenfant JM, Altfeld M. CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods. 2004;294:15-22. 23. Uhrberg M. The CD107 mobilization assay: viable isolation and immunotherapeutic potential of tumor-cytolytic NK cells. Leukemia. 2005;19:707-709. 24. Pascal V, Schleinitz N, Brunet C, et al. Comparative analysis of NK cell subset distribution in normal and lymphoproliferative disease of granular lymphocyte conditions. Eur J Immunol. 2004;34:2930-2940. 25. Toneva M, Lepage V, Lafay G, et al. Genomic diversity of natural killer cell receptor genes in three populations. Tissue Antigens. 2001;57:358-362. 26. Hsu KC, Liu XR, Selvakumar A, Mickelson E, O'Reilly RJ, Dupont B. Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes, each with multiple subsets. J Immunol. 2002;169:5118-5129. 27. Montoya CJ, Velilla PA, Chougnet C, Landay AL, Rugeles MT. Increased IFN-gamma production by NK and CD3+/CD56+ cells in sexually HIV-1-exposed but uninfected individuals. Clin Immunol. 2006. 28. Alter G, Malenfant JM, Delabre RM, et al. Increased natural killer cell activity in viremic HIV-1 infection. J Immunol. 2004;173:5305-5311. 29. Jacobs R, Weber K, Wendt K, Heiken H, Schmidt RE. Altered coexpression of lectin-like receptors CD94 and CD161 on NK and T cells in HIV patients. J Clin Immunol. 2004;24:281-286. 30. Ljutic B, Carlyle JR, Filipp D, Nakagawa R, Julius M, Zuniga-Pflucker JC. Functional requirements for signaling through the stimulatory and inhibitory mouse NKR-P1 (CD161) NK cell receptors. J Immunol. 2005;174:4789-4796. 31. Rosen DB, Bettadapura J, Alsharifi M, Mathew PA, Warren HS, Lanier LL. Cutting Edge: Lectin-Like Transcript-1 Is a Ligand for the Inhibitory Human NKR-P1A Receptor. J Immunol. 2005;175:7796-7799. 32. Aldemir H, Prod'homme V, Dumaurier MJ, et al. Cutting edge: lectin-like transcript 1 is a ligand for the CD161 receptor. J Immunol. 2005;175:7791-7795. 33. Azzoni L, Zatsepina O, Abebe B, Bennett IM, Kanakaraj P, Perussia B. Differential transcriptional regulation of CD161 and a novel gene, 197/15a, by IL-2, IL-15, and IL-12 in NK and T cells. J Immunol. 1998;161:3493-3500. 34. Bonaparte MI, Barker E. Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules. Blood. 2004;104:2087-2094. 35. Mela CM, Burton CT, Imami N, et al. Switch from inhibitory to activating NKG2 receptor expression in HIV-1 infection: lack of reversion with highly active antiretroviral therapy. Aids. 2005;19:1761-1769. 36. Khakoo SI, Thio CL, Martin MP, et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science. 2004;305:872-874. 37. Carr WH, Rosen DB, Arase H, Nixon DF, Michaelsson J, Lanier LL. Cutting Edge: KIR3DS1, a Gene Implicated in Resistance to Progression to AIDS, Encodes a DAP12-

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Associated Receptor Expressed on NK Cells That Triggers NK Cell Activation. J Immunol. 2007;178:647-651. 38. Gaudieri S, DeSantis D, McKinnon E, et al. Killer immunoglobulin-like receptors and HLA act both independently and synergistically to modify HIV disease progression. Genes Immun. 2005;6:683-690. 39. O'Connor G M, Guinan KJ, Cunningham RT, Middleton D, Parham P, Gardiner CM. Functional Polymorphism of the KIR3DL1/S1 Receptor on Human NK Cells. J Immunol. 2007;178:235-241. 40. Andre P, Brunet C, Guia S, et al. Differential regulation of killer cell Ig-like receptors and CD94 lectin-like dimers on NK and T lymphocytes from HIV-1-infected individuals. Eur J Immunol. 1999;29:1076-1085. 41. Fauci AS, Mavilio D, Kottilil S. NK cells in HIV infection: paradigm for protection or targets for ambush. Nat Rev Immunol. 2005;5:835-843. 42. Tran HK, Chartier L, Troung LX, et al. Systemic immune activation in HIV-1-exposed uninfected Vietnamese intravascular drug users. AIDS Res Hum Retroviruses. 2006;22:255-261. 43. Follezou JY, Lan NY, Lien TX, et al. Clinical and biological characteristics of human immunodeficiency virus-infected and uninfected intravascular drug users in Ho Chi Minh City, Vietnam. Am J Trop Med Hyg. 1999;61:420-424.

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Table 1: Genomic, transcriptional and phenotypic analysis of the KIR repertoire in EUs. HLA-B and -C typing was performed and grouped as HLA-C1 (Asn80), HLA-C2 (Lys80) and Bw4/Bw6 groups, according to KIR recognition pattern. Real-time PCR evaluation of each KIR transcript levels was performed in 25 EUs and associated to KIR genotyping in 14 EUs When KIR transcripts were detected (+), the ratio between activating and inhibitory counterparts was indicated. Phenotypic expression of KIR receptors is indicated as the percentage of KIR+ cells among CD3-CD56+ NK cells using EB6+, GL183+, Z27.3.7+, and FES172 mAbs. Data from exposed uninfected individuals (EU) were analyzed in reference to median values and 25th-75th percentiles in low-risk Vietnamese unexposed control blood donors (C) and HIV-infected individuals (HIV, lower panel)

Genotype 2DS1/2DL1

Transcripts 2DS1/2DL1 ratio

Phenotype % EB6+

Genotype 2DS2/2DL2/2DL3

Transcripts 2DS2/2DL2/2DL3

Phenotype % GL183+

Genotype 3DS1/3DL1

Transcripts 3DS1/3DL1 ratio

Phenotype % Z27.3.7+

Genotype 2DS4

Transcripts 2DS4/1D

Phenotype % FES172+

1 C1 Bw4 Bw6 -/+ -/+ 15 -/-/+ -/-/+ 24 -/+ -/+ 30 + +/- 532 C1 Bw4 Bw6 -/+ -/+ 21 -/-/+ -/-/+ 57 -/+ -/+ 7 + +/- 813 C1 Bw4 Bw6 nd -/+ nd nd -/-/+ nd nd -/+ nd nd +/- nd4 C1 Bw4 Bw6 -/+ -/+ 6 +/+/+ +/+/+ 63 -/+ -/+ 13 + -/+ 385 C1 Bw6 -/+ -/+ 32 -/-/+ -/-/+ 68 -/+ -/- 0 + -/- 06 C1 Bw6 +/+ +/+ 2.8 29 -/-/+ -/-/+ 26 +/+ +/+ 28 6 + -/+ 07 C1 Bw6 +/+ +/+ 61 4 +/+/+ +/+/+ nd +/+ +/+ 59 1 + -/+ 08 C1 Bw6 nd +/+ 0.9 8 nd +/+/+ 33 nd -/+ 22 nd +/+ 459 C1 Bw6 nd +/+ 3.7 nd nd -/-/+ nd nd +/+ 1.3 nd nd -/+ nd

10 C1 C2 Bw4 Bw6 +/+ +/- 13 +/+/+ +/+/+ 67 +/+ +/- 6 + +/+ 2411 C1 C2 Bw4 Bw6 nd +/+ 4.0 50 nd -/-/+ 54 nd +/+ 3.0 27 nd +/+ 4412 C1 C2 Bw4 Bw6 +/+ +/+ 1.0 83 +/+/+ +/+/+ 6 +/+ +/+ 1.9 6 + +/- 3713 C1 C2 Bw4 Bw6 +/+ +/+ 2.3 21 -/-/+ -/-/+ 36 +/+ +/+ 0.9 26 + -/+ 014 C1 C2 Bw4 Bw6 nd -/+ nd nd -/-/+ nd nd -/- nd nd +/+ nd15 C1 C2 Bw6 -/+ -/- 10 +/+/+ -/+/+ 41 +/+ +/- nd + +/+ 1616 C1 C2 Bw6 +/+ +/+ 1.0 38 -/-/+ -/-/+ 32 +/+ +/+ 28 8 + -/+ 017 C1 C2 Bw6 -/+ -/+ 9 +/+/+ +/+/+ 12 -/+ -/+ 5 + +/- 3018 C1 C2 Bw6 +/+ +/+ 0.1 47 -/-/+ -/-/+ 63 -/+ -/+ 17 + +/+ 2219 C2 Bw4 Bw6 nd -/+ nd nd -/-/+ 31 nd -/+ nd nd +/- nd20 C2 Bw6 -/+ -/+ nd -/-/+ -/-/+ nd -/+ -/+ nd + -/+ nd21 nd nd nd +/+ 1.0 25 nd -/-/+ 54 nd +/+ 8.1 10 nd +/- 4022 nd nd nd +/+ 3.45 31 nd +/+/+ 46 nd +/+ 3.8 21 nd +/- 4523 nd nd nd -/+ 5 nd +/+/+ 19 nd -/+ 1 nd -/+ 024 nd nd nd +/+ 47 20 nd +/+/+ 40 nd +/ni 8 nd -/- nd25 nd nd nd -/+ nd nd -/-/+ 30 nd -/+ nd nd -/+ nd

HIV - - -1.4

(1.4-1.4)11.3

(7.6-22.5) - -

28.4 (19.5-44.1)

-2.1

(2.1-2.1)11.3

(6.3-15.7) - -

33.7 (18.1-37.7)

EU - - -1.7

(0.5-3.6)15

(9-31) - -

36 (23-56.7)

-4.7

(1.5-28.0)8.6

(6.9-17.5) - -

30.5 (20.9-42.0)

C - - -4.1

(3.1-4.8)14.5

(7-26) - -

35.2 (20-48.3)

-3.0

(2.0-10.0)12

(8.8-21.5) - -

32.6 (21.7-65.0)

KIR2DS2/2DL2/2DL3 KIR3DS1/KIR3DL1 KIR2DS4EU HLA-C HLA-B

KIR2DS1/KIR2DL1

F

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Figure Legends

Figure 1: Genomic and real time PCR transcriptional analysis of the KIR repertoire.

A. The size of the NK cell compartment was analyzed by flow cytometry (% CD3-

CD56+CD16+ cells) in exposed HIV-infected IDU (HIV �) exposed HIV-uninfected IDU

(EU �) and unexposed control blood donors (C �).

B. The frequency of KIR transcript detection was analyzed in 17 HIV-infected, 25 EUs and 21

unexposed controls. Analysis of genomic KIR frequencies was conducted on a subgroup of 12

unexposed controls (and compared to genomic KIR frequencies reported by Toneva et al in

59 Vietnamese subjects25). HIV+ individuals show lower KIR transcript frequencies for

KIR3DS1 (p<0.10) and KIR3DL1 (p<0.001). Genomic analysis confirmed the weak KIR3DS1

frequency in HIV+ (18%). Dissociation between KIR3DL1 gene and transcript detection was

detected in 3 out of 14 EU tested (21.4%) and 3 out of 8 HIV (37.5%) individuals, but was

never observed in tested controls (n=12). Expressed KIR2DS4 transcript frequencies refer to

transcription alleles encoding the expressed KIR2DS4 receptor (exclusive of individuals only

expressing the KIR1D null allele). Level of significance is indicated as follow: * p<0.05, **

p<0.01, *** p<0.001.

C. Real time PCR analysis of altered KIR transcript patterns in HIV and EUs. KIR2DL4,

KIR3DL2, KIR2DL3 and KIR3DL1 mRNA transcript levels are represented as relative copy

numbers per 106 GAPDH transcripts. KIR2DL3 transcript levels where examined according to

the alternative KIR2DL3+KIR2DS2-KIR2DL2- or KIR2DL3+KIR2DS2+KIR2DL2+ genomic

gene combinations (middle left) and also plotted according to the size of the phenotypically

defined GL183+ NK cell subset (middle right). High percentage of GL183+ NK cells (> 50%

of GL183+ NK cells within CD3-CD56+ NK cells) identified a specific subgroup of

KIR2DL3+KIR2DS2-KIR2DL2- EU individuals who exhibit higher KIR2DL3 transcript levels

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in the absence of KIR2DS2/KIR2DL2 transcription. Activating KIR3DS1/inhibitory KIR3DL1

transcript ratio is represented (bottom right).

Figure 2: Flow cytometer analysis of NKR expression on CD3-CD56+ NK cells.

A. The size of NKR+ NK cell subsets expressing NKp30, NKp46, CD161, NKG2A and

NKG2C, CD69, CD85j were evaluated after gating of positive CD3-CD56+ NK cells in

exposed HIV-infected IDU (HIV �), exposed HIV-uninfected IDU (EU �), and unexposed

control blood donors (C �, 25th percentile, median, 75th percentile). NKp30 and NKp46 were

expressed on all NK cells (100% CD3-CD56+), and comparison of expression levels where

thus analysed by analysis of mean fluorescence intensity (mfi). NK cell surface expression of

NKG2A inhibitory subunit of the CD94/NKG2A heterodimeric receptor was inversely

correlated to that of the stimulatory NKG2C subunit among CD3-CD56+ NK cells in EU

subjects (Spearman’s correlation test), while correlation was non significant in C and HIV

groups.

B. Representative flow cytometer analyses of NKR staining in exposed HIV-infected IDU

(HIV), exposed uninfected IDU (EU) and unexposed controls (C) (white: isotypic control

staining, black, NKR specific staining).

Figure 3: Constitutive and inducible activation of NK cells is revealed by the CD107

degranulation assay.

A. Comparative analysis of CD107a expression on CD3-CD56+ NK cells was performed in

the three study groups, in the absence of stimulation (unstim) and upon K562 target cells

stimulation (K562). Boxes represent CD3-CD56+ cells expressing CD107a in exposed HIV-

infected IDU (HIV), exposed HIV-uninfected IDU (EU), and unexposed control donors (C), y

axis refer to 25th percentile, median, 75th percentile values.

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B. Comparative analysis of the mean fluorescence intensity (mfi) of CD107a staining among

CD107+CD3-CD56+ NK cells in exposed HIV-infected IDU (HIV), exposed uninfected IDU

(EU) and controls (C) (white: isotypic control staining, black, NKR specific staining). In the

absence of stimulation (unstim) and upon stimulation with K562 target cells (K562). Boxes

represent the median mfi of CD107a staining among CD3-CD56+ NK cells (25th percentile,

median, 75th percentile).

C. Examples of flow cytometer analyses of CD107a staining on CD3-CD56+ NK cells in the

absence of stimulation (white: isotypic control staining, black, NKR specific staining).

Figure 4: Analysis of CD161 expression by the activated CD107a+CD3-CD56+ NK cell

subset.

A. Cell surface expression of CD161 was evaluated by four-color flow cytometry after gating

on CD107a+CD3-CD56+ NK cells. The percentage of CD161+ stained cells was analyzed

within CD107a+CD3-CD56+ NK cells in absence of stimulation (unstim) and after 3 hours of

K562 cell stimulation (K562) in exposed HIV-infected IDUs (HIV), exposed uninfected IDUs

(EU) and unexposed controls (C). Y-axis values boxed represent 25th percentile, median and

75th percentile.

B. Representative flow cytometer analysis of % and mean fluorescence intensity (mfi) of

CD161 staining among the CD107a+CD3-CD56+ NK cell subset before exogenous stimulation

(white: isotypic control staining, black, CD161 specific staining).

C. Mean fluorescence intensity (mfi) of CD161 staining among the CD161+ unstimulated

CD107a+CD3-CD56+ NK cell subset. Y-axis values boxed represent 25th percentile, median

and 75th percentile.

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D. Evaluation of mean fluorescence intensity of CD161 expression within the CD107apositive

and CD107anegative CD3-CD56+NK cell subsets after K562 cell stimulation. Y-axis values

boxed represent 25th percentile, median and 75th percentile.

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HIV EU C0

10

20

30

40

50p<0.05

p<0.05

HIV EU C100

1000

10000

100000p<0.05

p<0.05

HIV EU C100

1000

10000

100000

1000000p<0.05

p<0.05

50000

150000

250000

350000

450000

550000p<0.05

2DS2/2DL2/2DL3Genotype

--+ +++HIV

--+ +++EU

--+ +++C

p<0.05 p<0.05

A B

C

0 10 20 30 40 50 60 70 801000

10000

100000

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HIVEU

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EU 2DL3+ only(2DS2-2DL2-)

(CD158b1/b2/j) cell surface phenotype% GL183+ on CD3-CD56+NK Cells

HIV EU C100

1000

10000

100000

HIV EU C0

10

20

30

40

50

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For personal use only. by guest on December 30, 2011. bloodjournal.hematologylibrary.orgFrom

B

84%4.24 mfi

NKp30

NKp46

HIV EU C

95%6.95mfi

38%1.55 mfi

mfi0.47

mfi1.29

mfi1.50

mfi0.70

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NKG2A

NKG2C

30%4.25 mfi

17%8.0 mfi

65%8.4mfi

10%2.08 mfi

50%2.61mfi

15%2.48 mfi

CD69

101 102 10100 101 102 10100101 102 103100 101 102 103100101 102 103100 101 102 103100

CD85j

42%2.84 mfi

41%2.16mfi

32 %1.96 mfi

10%2.26 mfi

18%1.72mfi

1.4%1.71 mfi

CD161HIV EU C0.00

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HIV EU C0

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0 10 20 30 40 50 60 70 80 90 1000

10

20

30

40

50

60

70

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p<0.05r = - 0.59

EU

% NKG2A+

on CD3-CD56+ cells

A

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bloodjournal.hematologylibrary.org

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unstim K562 unstim K562 unstim K562 0

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