Mechanisms of allergy and clinical immunology

CD94/NKG2C is a killer effector molecule in patients withStevens-Johnson syndrome and toxic epidermal necrolysis

Esther Morel, PhD,a Salvador Escamochero, BS,a Rosario Cabanas, MD, PhD,b Rosa Dıaz, MD, PhD,c Ana Fiandor, MD,b

and Teresa Bellon, PhDa Madrid, Spain

Background: Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are severe, bullous cutaneous diseaseswith uncertain pathogenesis, although cytotoxic T cells seem tobe involved. Natural killer (NK)–like activity has been found inblister infiltrates. Cytotoxic T lymphocytes (CTLs) with NK-likeactivity (NK-CTLs) have been shown to express T-cell receptorsrestricted by the HLA-Ib molecule HLA-E. Alternatively, theHLA-E–specific activating receptor CD94/NKG2C can triggerT-cell receptor–independent cytotoxicity in CTLs.Objective: Our aim was to test whether HLA-E expressionsensitizes keratinocytes to killing by CTLs with NK-like activityand to explore the expression of activating receptors specific forHLA-E in blister cytotoxic lymphocytes.Methods: We used flow cytometry and immunohistochemistryto analyze HLA-E expression in keratinocytes from affectedskin in patients with SJS, TEN, and other less severe drug-induced exanthemas. The expression of CD94/NKG2C wasanalyzed by means of flow cytometry in PBMCs and blister cellsfrom patients. PBMCs and blister cells were analyzed for theirability to kill HLA-E–expressing cells. Involvement of CD94/NKG2C in triggering degranulation of cytolytic cells wasexplored by means of CD107a mobilization assays and standardcytotoxicity chromium release assays.Results: We found that keratinocytes from affected skinexpressed HLA-E and that cell-surface HLA-E sensitizeskeratinocytes to killing by CD94/NKG2C1 CTLs. Frequenciesof CD94/NKG2C1 peripheral blood T and NK cells wereincreased in patients with SJS and TEN during the acute phase.Moreover, activated blister T and NK lymphocytes expressedCD94/NKG2C and were able to degranulate in response toHLA-E1 cells in an NKG2C-dependent manner.Conclusion: CD94/NKG2C might be involved in triggeringcytotoxic lymphocytes in patients with SJS and TEN. (J AllergyClin Immunol 2010;125:703-10.)

From athe Research Unit, Hospital Universitario La Paz–FIBHULP and bthe Allergy

Service and cthe Dermatology Service, Hospital Universitario La Paz.

Supported by grant PI 06/0441 from the Ministerio de Sanidad/ISCIII, Spain, to T.B..

E.M. is the recipient of a fellowship from the FIBHULP.

Disclosure of potential conflict of interest: The authors have declared that they have no

conflict of interest.

Received for publication March 22, 2009; revised September 9, 2009; accepted for

publication October 19, 2009.

Available online February 4, 2010.

Reprint requests: Teresa Bellon, PhD, Research Unit, Hospital Universitario ‘‘La Paz,’’

P8 Castellana 261, 28046 Madrid, Spain. E-mail: tbellon.hulp@salud.madrid.org.

0091-6749/$36.00

� 2010 American Academy of Allergy, Asthma & Immunology

doi:10.1016/j.jaci.2009.10.030

Key words: Stevens-Johnson syndrome, toxic epidermal necrolysis,drug allergy, NK-CTLs, CD94/NKG2C, HLA-E

Cutaneous reactions are the most frequent manifestations ofdelayed drug-induced hypersensitivity.1 They comprise a broadspectrum of clinical features spanning benign diseases, such asmaculopapular exanthema (MPE), to life-threatening severe reac-tions. Among them, Stevens-Johnson syndrome (SJS) and toxicepidermal necrolysis (TEN) are the most severe forms of drug-induced skin diseases and are now considered variants of thesame disease.2 SJS and TEN are rare diseases with a prevalenceof 1 to 2 cases per million persons per year in white subjectsand a mortality rate ranging from 2% to 10% in patients withSJS and 30% in patients with TEN. The most common featureof these diseases is the formation of subepidermal blisters anddetachment of the epidermis, which appears as scalded skin.Necrosis of the full thickness of the epidermis is the pathogno-monic finding in this entity.3 Blisters under the necrotic epidermiscontain T lymphocytes, which can be regarded as the effectors ofthe immune reaction. T cells within blisters showed high expres-sion of CD56,4 which has been associated with the acquisition ofcytolytic effector functions by cytotoxic T lymphocytes (CTLs).5

Moreover, these cells express high levels of natural killer (NK)cell receptors (NKRs) and showed NK-like cytotoxic activity.6

T cells exhibiting NK-like activity have been named NK-CTLsand are characterized as T cells expressing CD56 and NKRs.7 Ithas been reported that T-cell receptors (TCRs) from NK-CTLsare HLA-E restricted, and therefore these cells kill targets throughthe recognition of the nonclassical MHC class Ib molecule HLA-E by specific TCRs.7,8 Additionally, two HLA-E–specific NKRs(CD94/NKG2A and CD94/NKG2C) have been identified in hu-man subjects.9 Both are expressed in subpopulations of NK andT cells. These heterodimers belong to the C-type lectin familyand consist of an invariant CD94 chain covalently linked toNKG2A or NKG2C. Although the extracellular regions ofNKG2A and NKG2C are highly homologous, the transmembraneand intracytoplasmic domains define opposite functions. CD94/NKG2A constitutes an inhibitory receptor because of the pres-ence of two immunotyrosine-based inhibitory motifs in theNKG2A cytoplasmic tail. However, CD94/NKG2C also consti-tutes an activating receptor because NKG2C contains a chargedresidue in its transmembrane region that allows its associationwith the immunoreceptor tyrosine-based activation motif–containing adaptor molecule DNAX activating protein of 12kDa (DAP12).10 It has recently been reported that CTLs mightuse CD94/NKG2C to kill HLA-E–expressing cells.11,12

Because blister lymphocytes express cell-surface moleculessimilar to those described for NK-CTLs and are endowed with

703

TABLE I. Expression of cell-surface receptors on T lymphocytes

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704 MOREL ET AL

from peripheral blood

Abbreviations used

Cell-surface Patients with Patients Healthy

BFC: B lister fluid cell

receptor TEN/SJS with MPE donors

CTL: C ytotoxic T lymphocyte

FasL: F

as ligand CD8 FITC: F luorescein isothiocyanate

Percent* 43.4 6 2.5 39 6 1.7 34.9 6 1

KIR: K iller immunoglobulin-like receptor N� 16 38 56 MPE: M aculopapular exanthema P value� .003 .04 NK: N atural killer

ILT2/CD85j

NKR: N atural killer receptor Percent 17.6 6 5.1 17 6 2.1 7.1 6 0.7 PE: P hycoerythrin N 14 34 49 rh: R ecombinant human P value .02 .0001 SJS: S tevens-Johnson syndrome

CD94

TCR: T -cell receptor Percent 11.5 6 2.6 11.2 6 1 8.6 6 0.6 TEN: T oxic epidermal necrolysis N 10 36 56

P value .37 .18

KIR2DL2/L3/S2

Percent 10 6 3 5.7 6 0.8 3.3 6 0.4

N 14 35 43

NK-like activity, we hypothesized that they could also beactivated by HLA-E expressed on target cells.

P value .007 .002

KIR2DL1/S1/S4

Percent 3.2 6 0.8 3.5 6 0.5 2.9 6 0.3

N 14 32 53

P value .57 .31

METHODSA list of materials and reagents used in this study is found in this article’s

Methods section in the Online Repository available at www.jacionline.org.

KIR3DL1/L2/2DS4

Percent 2.8 6 0.5 4.4 6 0.9 2.3 6 0.4

N 15 35 54

P value .22 .05

KIR3DL1

Percent 4.4 6 2.4 2.4 6 0.7 2.3 6 0.3

N 16 32 50

P value .98 .38

*CD81, ILT2/CD85j1, CD941, and KIR1 cell subsets were defined by means of flow

cytometry in gated CD31 lymphocytes. Data are expressed as means 6 SEMs.

�Number of samples.

�Statistical analysis of PBMCs from patients with TEN/SJS and MPE in the acute

phase versus healthy donors (Mann-Whitney U test).

Human samplesPatients meeting the criteria for drug-induced delayed generalized cutane-

ous reactions1,13 were evaluated in the Allergy Service of Hospital La Paz. All

patients had negative serologic results for HIV or mycoplasma infection, no

hematologic disorders were involved, and none of the patients were receiving

corticosteroids before blood sampling. Fifteen patients with TEN or SJS and

38 patients with MPE associated to various drugs participated in the study (see

Tables E1 and E2 in this article’s Online Repository at www.jacionline.org).

Peripheral blood was drawn from healthy donors or from patients at the mo-

ment of admission to the hospital (acute samples) or on complete remission

of the clinical symptoms, typically at least 1 month after hospital discharge

in patients with SJS/TEN (resolution samples). PBMCs were isolated by

means of Ficoll/Hypaque (GE Healthcare, Uppsala, Sweden) density gradient

centrifugation of blood samples. Blister fluids were obtained from tense blis-

ters by means of puncture aspiration into a syringe. Blister fluid cells (BFCs)

were collected by means of centrifugation for functional assays, and blister

fluids were harvested and frozen at 2808C until use.

Cell suspensions were obtained by using 2 steps of 20 minutes’ digestion

with 0.1% Trypsin and 5 mmol/L EDTA of skin biopsy specimens. Cultures of

primary human keratinocytes were generated as previously described14 and

are explained in the Methods section in this article’s Online Repository.

Informed consent was obtained from all participants or their legal

representatives. The study was approved by the Ethics Committee of Hospital

La Paz and conducted according to Declaration of Helsinki principles.

Cell lines, mAbs, and flow cytometryCell lines, mAbs, and flow cytometry are detailed in the Methods section in

this article’s Online Repository.

ImmunohistochemistryImmunohistochemistry was performed as described in the Methods section

in this article’s Online Repository by using the anti–HLA-E–specific mAb

MEM-E/02.

Western blottingProtein content in sera and blister fluids was quantified, and equal amounts

of protein (50 mg) were resolved by 10% SDS-PAGE and transferred onto

polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules,

Calif). Equal volumes of culture supernatants from 721.221 and 221.AEH

cell lines (20 mL) were loaded as control samples. Filters were probed with

MEM-E/02 mAb and anti-mouse IgG horseradish peroxidase–linked anti-

bodies (Chemicon International, Temecula Calif). Immunoreactivity was

detected with the ECL Advance Western Blotting detection kit (GE

Healthcare).

Isolation, magnetic separation, and cloning of

CD561 lymphocytesPBMCs from healthy donors were incubated in RPMI-1640 with 10% heat-

inactivated FCS for collecting nonadherent cells. Afterward, CD561 cells

were isolated by means of positive selection with CD56 magnetic microbeads

(Miltenyi Biotec, Bergisch Gladbach, Germany). CD561 purified cells were

seeded under limiting dilution conditions in round-bottom microtiter plates

in RPMI-1640 with 20% heat-inactivated FCS, irradiated (6000 rads) alloge-

neic PBMCs (3 3 104 cells/well), 20 U/mL recombinant human (rh) IL-2

(Peprotech, London, United Kingdom), and 1 mg/mL PHA (Murex, London,

United Kingdom). The generated clones were analyzed by means of flow

cytometry, and CD31CD81CD561 clones were selected for subsequent

cytotoxicity assays.

Cytotoxicity assaysCell-mediated cytotoxicity was measured in a standard 4-hour 51Cr release

assay (PerkinElmer, Boston, Mass) against HaCaT cells and human primary

FIG 1. HLA-E expression in affected skin. A, Flow cytometric analysis of HLA-I (solid histograms) and HLA-E

(open histograms) expression in keratinocytes from a representative patient with MPE and a healthy donor

(HD). B, Mean fluorescence intensity (MFI) of HLA-I and HLA-E expression in keratinocytes from 5 healthy

donors (HD) and 8 patients (P). Horizontal bars correspond to the mean values. C, Immunoperoxidase stain-

ing of HLA-E in skin biopsy specimens from 3 patients. Original magnification 2003 (panels a-f) and 4003

(panels g-h). Arrowheads in panel h show specific staining in keratinocytes within necrotic skin. D, Immu-

noblot analysis of soluble HLA-E (sHLA-E) in fluids and sera from acute or resolution samples in 3 represen-

tative patients with SJS/TEN, healthy donors, and patients with mechanical trauma (MT). Culture

supernatants from the HLA-I2 cell line 721.221 and its HLA-E transfectant, 221.AEH, were used as negative

and positive controls.

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MOREL ET AL 705

keratinocytes pretreated or not with rhIFN-g and 721.221 and 221.AEH cell

lines. Assays were conducted in RPMI plus 10% heat-inactivated FCS in

the presence or absence of saturating concentrations (10 mg/mL) of the

specific mAbs 3D12, W6/32, and/or anti-NKG2C. CD561CD81 T-cell

clones, PBMCs, and BFCs were used as effector cells at a 50:1 (PBMCs) or

20:1 (BFCs) effector/target ratio. Assays were conducted in triplicates by

using U-bottom 96-well microtiter plates. Specific lysis was calculated as

follows:

100 3 ðSpecific release ½cpm� � Spontaneous release ½cpm�=Maximum release ½cpm� 2 Spontaneous release ½cpm�Þ:

In every case spontaneous release was less than 20% of the maximum lysis.

CD107a mobilization assaysBFCs or PBMCs from patients’ acute samples were cultured or not at a

2:1 (efector:target) ratio for 4 hours with the 721.221 and 221.AEH cell

lines or with autologous keratinocytes, pretreated or not with 10 ng/mL

rhIFN-g for 48 hours, and in the presence or absence of specific mAbs anti-

NKG2C (10 mg/mL). Phycoerythrin (PE)–conjugated anti-human CD107a

and Monensin A (2 mmol/L; Sigma-Aldrich, Lyon, France) were added

during the coculture period. After this time, cells were collected and

stained with the specific mAbs CD3–peridinin-chlorophyll-protein (PerCP),

CD8–fluorescein isothiocyanate (FITC), or anti-NKG2C. A secondary

FITC-conjugated rabbit anti-mouse F(ab9)2 was used for detection of

NKG2C. Assays were performed in triplicates. CD107a1 cells were ana-

lyzed by means of flow cytometry on selected populations within the lym-

phocyte gate.

Statistical analysisStatistical analyses used in this study are described in the Methods section

in this article’s Online Repository.

RESULTS

Expression of NKRs in lymphocytes from patients

with SJS and TENTo confirm previously reported data, we performed a flow

cytometric analysis of cell-surface expression of various NKreceptors in BFCs of patients with SJS/TEN. Additionally, thephenotype of peripheral blood lymphocytes was studied inPBMCs drawn simultaneously to the blister cells (acute samples)and on complete remission of the clinical symptoms (resolutionsamples). Most blister lymphocytes were CD81 and wereenriched in cells expressing CD56, CD94, and killer immuno-globulin-like receptors (KIRs). Moreover, the proportion of cellsexpressing these receptors was increased in peripheral blood dur-ing the acute phase of SJS/TEN (see Fig E1 in this article’s OnlineRepository at www.jacionline.org). Table I shows significantly in-creased CD81 T cells in patients with SJS/TEN compared withnumbers seen in healthy donors, which is in agreement with pre-vious reports.15,16 The expression of other NKRs, such as ILT2/CD85j and KIR2DL2/L3/S2, was also significantly increased inpatients with drug-induced hypersensitivity reactions, whereas

FIG 2. Cytotoxic lymphocytes are activated by HLA-E in peripheral blood

and blister fluid from patients with SJS/TEN. A, 51Cr release assays show-

ing lysis of PBMCs from healthy donors and patients with MPE and SJS/

TEN against the 721.221 and 221.AEH cell lines. Means and SEMs of inde-

pendent experiments are shown. B and C, BFCs from patients with SJS/

TEN were used as effector cells in 51Cr release assays. Fig 2, B, Mean values

and SEMs of percentages of lysis against 721.221 and 221.AEH cells. Fig 2,

C, Lysis of primary allogenic keratinocytes (KC) pretreated or not with IFN-g

by BFCs from 2 representative patients.

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706 MOREL ET AL

no significant differences were found in the percentages of cellsexpressing CD94, KIR2DL1/S1/S4, and KIR3D receptors.

High levels of soluble Fas ligand (FasL) were found in blisterfluids from patients with SJS/TEN. IFN-g levels were signifi-cantly higher in sera from patients, and slightly higher concen-trations were detected in blisters (see Fig E2 in this article’sOnline Repository at www.jacionline.org).

HLA-E is expressed in keratinocytes from affected

skinAnalysis of cell suspensions from affected skin biopsy

specimens revealed increased HLA-I and HLA-E cell-surfaceexpression in CD452 cells (Fig 1, A and B). These data sug-gested an upregulation of HLA-E cell-surface expression in ep-idermal keratinocytes from patients with drug-induced delayedhypersensitivity reactions. This was confirmed by means of im-munohistochemistry in skin biopsy specimens of patients withMPE and TEN (Fig 1, C). Moreover, although soluble HLA-E was not found in sera from patients and healthy donors orin blister fluids from mechanical trauma, immunoblot assaysdemonstrated that soluble HLA-E is released into the blisterfluid in patients with SJS/TEN (Fig 1, D), which further sup-ports the expression of HLA-E by epidermal cells in thesepatients.

HLA-E cell-surface expression in human

keratinocytes sensitizes them to killing by CD561

CTLsFlow cytometric analysis revealed no detectable expression

of HLA-E either in the cell membrane of in vitro cultured pri-mary keratinocytes or in the human keratinocyte cell line

HaCaT. However, IFN-g stimulation induced cell-surface ex-pression of HLA-E in primary cultures of human keratinocytes,as well as in HaCaT cells (Fig E3, A, in this article’s Online Re-pository at www.jacionline.org). CD561CD81 T-cell clonesgenerated from healthy donors and selected based on increasedcytotoxicity against the HLA-E–transfected cell line(221.AEH)17 compared with the HLA-I–deficient parental cellline 721.221 were also able to kill HaCaT cells previously stim-ulated with IFN-g (see Fig E3, B) in an HLA-E–dependentmanner, as demonstrated when cytotoxicity assays were per-formed in the presence of the blocking antibodies anti-HLAclass I or anti-HLA-E (Fig E3, C). Altogether, these datashow that HLA-E cell-surface expression in keratinocytes sen-sitizes them to killing by CD561CD81 CTLs restricted or acti-vated by HLA-E.

Lymphocytes expressing HLA-E–specific activating

receptors are found in PBMCs from patients with

SJS and TENPBMCs from healthy donors and patients during the acute

phase of the disease were used as effector cells in 51Cr releasecytotoxicity assays against HLA-I–deficient and HLA-E1 cells.The expression of HLA-E in target cells significantly inhibitedcell lysis by PBMCs drawn from patients with MPE or healthydonors, which is in agreement with the high frequency of lympho-cytes expressing the HLA-E–specific inhibitory receptor CD94/NKG2A in healthy subjects.18 In contrast, PBMCs from patientswith SJS/TEN showed similar cytolytic activity against HLA-I–deficient and HLA-E1 targets (Fig 2, A). Because no differenceswere found in CD94/NKG2A expression in patients and healthydonors (see Fig E4 in this article’s Online Repository at www.jacionline.org), these results suggest an increased ratio of effectorcells expressing activating versus inhibitory receptors specific forHLA-E in peripheral blood from patients with SJS/TEN.Similarly, when BFCs were used as effector cells, we did notfind inhibition of the percentage of lysis by the expression ofHLA-E in targets (Fig 2, B). Moreover, BFCs showed increasedcytolytic activity against IFN-g–treated primary keratinocytes(Fig 2, C).

High frequencies of cytotoxic lymphocytes

expressing the HLA-E–specific activating receptor

CD94/NKG2C are found in patients with SJS/TENTwo activating receptors expressed in T cells, NK cells, or both

might be involved in the recognition of HLA-E in keratinocytes.HLA-E–restricted TCRs have been described in NK-CTLs.7,19

Additionally, it has recently been reported that CTLs might alsouse the activating NKR CD94/NKG2C to kill HLA-E1 targetcells.12,19 The availability of a specific mAb against CD94/NKG2C allowed us to study the expression of this receptor inPBMCs and compare it with its inhibitory counterpart, CD94/NKG2A, in the same donors. Although in healthy donors the pro-portion of CD94/NKG2A1 NK and T cells was found to be sig-nificantly higher than that of CD94/NKG2C1 NK and T cells,no differences were found in the frequencies of activating and in-hibitory receptors in patients with SJS/TEN. CD94/NKG2C,which is expressed at low frequency in T cells from healthy sub-jects (mean 6 SD, 2.6% 6 2.0%), was found on the cell surfaceof a substantial proportion of T cells from patients with SJS/TEN

FIG 3. CD94/NKG2C expression in lymphocytes or blasts from patients with SJS/TEN. A, Frequencies of

CD94/NKG2C1 cells in CD31CD81, CD31CD82, and CD32 peripheral blood lymphocytes from 3 representa-

tive acute samples (SJS8, SJS2, and TEN2) and 1 resolution sample (TEN2 resolution). B, Frequencies of

CD94/NKG2C1 cells in blister lymphocytes or blasts in 3 representative patients. PerCP, Peridinin-chloro-

phyll-protein.

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MOREL ET AL 707

(mean 6 SD, 4.7% 6 4.04%), although no statistical significancewas achieved (see Fig E4, A). Additionally, significantly higherfrequencies of CD94/NKG2C1CD32 NK cells were found inperipheral blood of patients with SJS/TEN during the acute phase(20.16% 6 17.6% in patients vs 2.15% 6 2.0% in healthy donors).As previously described,12 CD94/NKG2C was found mainly inthe CD81 subpopulation of CD31 lymphocytes (Fig 3, A). Nota-bly, after recovery, patients experienced a sharp decrease in thefrequency of peripheral CD94/NKG2C1CD81CD31 or CD94/NKG2C1CD32 lymphocytes (Fig 3, A, and Fig 4, B). Higherfrequencies of CD94/NKG2C1 cells were found also inCD31CD81 and CD32 cells from patients with SJS/TEN whencompared with those seen in patients with drug-induced MPE(see Fig E4, B).

The expression of CD94/NKG2C was also analyzed in BFCsfrom patients with bullous reactions. According to flow cyto-metric morphologic criteria (forward light scatter), an importantproportion of blasts was found within blisters. High frequenciesof CD94/NKG2C1 cells were detected in CD81CD31 cells, par-ticularly when blasts were analyzed. In addition to CD31CD81

cytotoxic T cells, CD94/NKG2C1CD32CD561 NK cells werefound in blister fluids from patients with SJS/TEN (Table II andFig 3, B).

In light of the high frequencies of CD94/NKG2C1CD81 T-cellblasts found in blisters, flow cytometric data were further analyzedto study the blast cell population in peripheral blood. Increasedfrequencies of CD94/NKG2C were detected within this fraction.Additionally, CD32CD561 NK cells were found as well withinthe blast region in some patients and were also enriched inCD94/NKG2C1 cells (see Fig E5 in this article’s Online Reposi-tory at www.jacionline.org and Fig 4, A). The population ofCD94/NKG2C1 blasts was dramatically decreased on completeremission of clinical symptoms (Fig 4, B). Altogether, these datashow that CD94/NKG2C is overexpressed in cytotoxic cellsand that CD94/NKG2C1 cytolytic cells are activated duringSJS/TEN.

CD94/NKG2C–HLA-E interactions trigger

cytotoxicity in peripheral blood and blister

lymphocytes from patients with SJS/TENCytotoxicity assays against 721.221 and 221.AEH cell lines

were conducted in which PBMCs from acute samples were usedas effector cells in the presence of blocking anti-NKG2C–specificantibodies. Although the cytotoxic activity against 721.221 cellswas not affected, lysis of 221.AEH cells was substantiallyreduced (Fig 5, A), indicating that CD94/NKG2C1 cells were re-sponsible for the increased cytolytic activity against HLA-E1

cells (see also Fig 2, A). Degranulation assays were performedand surface expression of CD107a was analyzed in specific sub-populations to further analyze the contribution of CD94/NKG2C1 CTLs and CD94/NKG2C1 NK cells. CD94/NKG2C1CD31 (T cells) and CD94/NKG2C1CD32 (NK cells)upregulated cell-surface CD107a on encounter with HLA-E1

cells (Fig 5, B), indicating a contribution of both cell lineagesto target lysis.

BFCs were also used as effector cells in CD107a degranulationassays against HLA-I2 and HLA-E1 cell lines. We found thatblister fluid T and NK cells were able to degranulate on exposureto HLA-E1 cells in a CD94/NKG2C-dependent manner becauseit was hampered in the presence of blocking NKG2C-specific an-tibodies (Fig 6, A and B). In line with these findings, degranulationof BFCs was also increased in a CD94/NKG2C-dependent way inthe presence of autologous keratinocytes previously stimulatedwith IFN-g to induce HLA-E cell-surface expression (Fig 6, C,upper graph). Interestingly, when CD32 cells were analyzed sep-arately, we found degranulation in the presence of unstimulatedautologous keratinocytes, which was further increased in anNKG2C-dependent manner if IFN-g–stimulated keratinocyteswere used as targets (Fig 6, C, lower graph). Altogether, thedata support an involvement of the activating receptor CD94/NKG2C, which is expressed in CTLs and NK cells, in the recog-nition and killing of HLA-E1 keratinocytes by blister cytotoxiclymphocytes in patients with SJS/TEN.

TABLE II. Expression of cell-surface receptors on lymphocytes

from blister fluids*

Blister

samples CD31CD81y

CD31CD81

CD94/NKG2C1z CD32CD561{ CD32CD94/NKG2C1{

SJS1 58.9 NT 41 NT

SJS2 46.2 11.4 34.8 7.2

SJS4 38.7 10.1 22 21.6

SJS5 83.3 32.3 24 23.1

SJS6 49.5 5.5 63.8 24.4

TEN1 80.4 2.3 6.5 5

TEN2 69.5 NT 81 NT

NT, Not tested.

*CD31CD81, CD31CD81CD94/NKG2C1, CD32CD561, and CD32CD94/

NKG2C1 blister lymphocytes were defined by means of flow cytometry. Data are

expressed as percentages of cell subpopulations.

�Analysis according to total CD31 lymphocytes.

�Analysis according to total CD31CD81 lymphocytes.

{Analysis according to total CD32 lymphocytes.

FIG 5. Peripheral blood NK and T lymphocytes from patients with SJS/TEN

recognize HLA-E through the activating receptor CD94/NKG2C. A, Cytotox-

icity mediated by PBMCs from patients’ acute samples and samples from

healthy donors against 721.221 and 221.AEH cell lines in the presence or ab-

sence of blocking anti-NKG2C mAbs. Means and SEMs of independent ex-

periments carried out with 3 patients with SJS are shown. B, CD107a

mobilization assays. Graphs show the percentage of CD107a1 cells in

CD31 or CD32 lymphocyte subpopulations expressing or not CD94/

NKG2C in a representative patient with SJS on coculture with 721.221

and 221.AEH cell lines.

FIG 4. CD94/NKG2C expression on peripheral blood lymphocytes and

blasts from patients with SJS/TEN. A, Frequencies of CD94/NKG2C1 cells

in peripheral blood lymphocytes or blast subpopulations in acute samples

from patients with SJS/TEN. B, Frequencies of CD94/NKG2C1 cells in

peripheral blood lymphocyte (L) or blast (B) subpopulations in acute and

resolution samples from 3 patients with SJS/TEN.

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DISCUSSIONSJS and TEN are severe hypersensitivity reactions to drugs

characterized by the widespread destruction of the epithelium ofthe skin and mucous membranes, which occurs through massiveapoptosis of keratinocytes.20 Formation of subepidermal bullae is

characteristic, and necrosis of the full thickness of the epidermisis the pathognomonic feature of SJS/TEN.3 Several mechanismshave been postulated to trigger this process.21 It has beenproposed that the death receptor Fas/CD95 plays a key role inthe apoptosis of keratinocytes that leads to epidermal necroly-sis.22 Furthermore, cell-free blister fluids are cytotoxic (unpub-lished data).23 Nonetheless, mononuclear cells are present inthe blister fluid, principally CD81 T lymphocytes, which are sup-posed to be the effector cells responsible for keratinocyte deatheither by means of direct killing or secretion of soluble cytotoxicproteins. Cytotoxic cells use at least 2 pathways to induce celldeath. A nonsecretory pathway involves the interaction of cell-surface receptor-ligand pairs. FasL on effector cells interactswith Fas/CD95 on target cells, inducing target cell death by acti-vating a caspase cascade. Additionally, a secretory pathwayinvolves the directional release of cytolytic granules containingperforin, granzymes, and granulysin. Recently, it has been sug-gested that granulysin can play an important role in inducingkeratinocyte death in patients with SJS and TEN,23 although othersoluble mediators, such as TNF-a,24 and soluble FasL,25 whichcan be released by cytotoxic lymphocytes on cell activation,might contribute to keratinocyte apoptosis. Nonetheless, therelease of cytolytic granules and soluble cytotoxic factors impliestriggering of effector cells through ligand recognition on targets.Published data suggest that blister T lymphocytes might exhibit adrug-specific MHC class I–restricted cytotoxicity against autolo-gous cells.26 Previously, it was published that blister T cellsexpressed NKRs and showed NK-like activity.6 The recognitionof the nonclassical HLA-Ib molecule HLA-E by specific TCRshas recently been reported to be the molecular mechanism respon-sible for NK-like activity in T cells, which was previously de-scribed as HLA-I unrestricted, and the term NK-CTL was

FIG 6. CD94/NKG2C-dependent degranulation in blister lymphocytes.

A and B, BFCs from patients SJS6 and SJS4 were cultured alone or with

721.221 and 221.AEH cells in the absence or presence of anti-NKG2C

mAbs. Fig 6, A, shows percentages of CD31CD81CD107a1 lymphocytes

in a representative experiment. Fig 6, B, CD32CD107a1 blister fluid lympho-

cytes in patient SJS6 after coculture in the absence or presence of 721.221

or 221.AEH cells and blocking anti-NKG2C mAbs. C, Induction of surface

CD107a in total and CD32 BFCs from patient SJS4 after coculture in the ab-

sence or presence of autologous keratinocytes (KC) pretreated or not with

IFN-g and with or without specific anti-NKG2C mAbs. Mean percentages

of CD107a1 lymphocytes in triplicate cultures are shown.

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proposed.7 An alternative pathway to activate CTL lysis of HLA-E1 targets through the activating receptor CD94/NKG2C wasalso described.12 In the present work we found an upregulationof HLA-E expression in keratinocytes and of CD94/NKG2C incytotoxic Tand NK cells from patients with SJS/TEN. In addition,we have demonstrated that HLA-E expression sensitizeskeratinocytes to killing by HLA-E–activated CTLs and BFCs.In T cells CD94 is acquired after antigen stimulation, andCD94/NKG2C expression is restricted to highly differentiated ef-fector cells.27,28 Functionally active CD94/NKG2C1 cells weredetected not only in blisters but also in peripheral blood frompatients during the acute phase. The simultaneous presence ofHLA-E–activated cytotoxic cells and HLA-Ia–restricted specificT cells is plausible and could explain our data and previousreports showing drug-independent lysis of allogenic keratino-cytes23 and drug-dependent lysis of autologous cells.26,29

Shedding of HLA-E has been described in IFN-g– and TNF-a–activated cells.30,31 The presence of soluble HLA-E withinblister fluids supports the expression of cell-surface HLA-E byactivated keratinocytes in patients with SJS/TEN. High levels ofIFN-g and TNF-a, which has been reported in blister fluidsfrom some patients,32 could stimulate surface expression ofHLA-E in keratinocytes from affected skin and the release of sol-uble molecules into the blister fluid.31 It has been proposed thatsoluble HLA-E can protect target cells from lysis through itsinteraction with the inhibitory receptor CD94/NKG2A.31 Like-wise, it could act as an agonist to activate cytotoxicity throughits interaction with activating receptors, such as CD94/NKG2C,HLA-E–restricted TCRs, or both. This issue deserves furtherresearch.

High concentrations of granulysin, a molecule containedwithin the cytolytic granules of CTLs and NK cells, have beenfound in blister fluids.23 The same authors reported high percent-ages of granulysin-expressing CTLs and NK cells in blisters.Consistent with these observations, our study supports a role forboth CTLs and NK cells in the induction of keratinocyte apoptosisin patients with SJS/TEN because we found that CD94/NKG2C1

blister NK cells, as well as CTLs, were able to degranulate in thepresence of HLA-E1 targets. Moreover, high expression of gran-ulysin has been reported in NKG2C1 CTLs.11

Similar to our results in patients with SJS/TEN, a predomi-nance of cells expressing CD94/NKG2C over CD94/NKG2A hasalso been found in HIV-infected subjects,33 and strikingly, the in-cidence of SJS/TEN augments in HIV-infected patients.3 On theother hand, the involvement of CD94/NKG2C in tissue damagehas been previously reported in patients with acute celiac disease,in whom CD94/NKG2C1 CTLs have been shown to kill HLA-E1

enterocytes,11 and expansion of CD94/NKG2C1 T cells and NK-CTLs have been detected in CMV-seropositive subjects,18,34 sug-gesting that these subpopulations might be involved in the immu-nopathology of various chronic infectious and inflammatorydiseases.11

In conclusion, our results suggest that the activating receptorCD94/NKG2C might be involved in triggering cytotoxic lym-phocytes in patients with SJS/TEN and that CD94/NKG2C–HLA-E ligation could be a mechanism involved in the physiopa-thology of these diseases. Targeting this pathway could be ofinterest in designing future treatments for these patients.

Clinical implications: Novel therapies targeting CD94/NKG2C–HLA-E interactions might be applicable for the treat-ment, prevention, or both of SJS and TEN.

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15. Correia O, Delgado L, Ramos JP, Resende C, Torrinha JA. Cutaneous T-cell

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METHODS

Cell linesThe HaCaT human keratinocyte cell line was grown in Dulbecco’s

modified Eagle medium with 5% (vol/vol) heat-inactivated FCS and in the

presence or absence of 2.5 ng/mL rhIFN-g (Peprotech) for 48 hours at 378C

and 5% CO2. The HLA class I–defective B-cell line 721.221 was grown in

RPMI-1640 medium supplemented with 5% heat-inactivated FCS. The

221.AEH cell line, a gift from Dr Geraghty (University of Washington, Seat-

tle, Wash), was cultured in the presence of 300 mg/mL hygromycin B (Calbio-

chem, La Jolla, Calif).

mAbsThe following mAbs were used: HP-1F7 (anti-HLA class I) and HP-3B1

(anti-CD94), kindly provided by Dr Lopez-Botet (Universitat Pompeu

Fabra, Barcelona, Spain), and 3D12 (anti-HLA-E), kindly provided by Dr

Geragthy (University of Washington). CD3–peridinin-chlorophyll-protein,

CD158b-FITC (anti-KIR2DS2, KIR2DL2, and KIR2DL3), HLA-DR–PE,

CD8-FITC, CD56-FITC, CD25-FITC, HLA-DR–FITC, and CD107a-PE

were purchased from BD Biosciences (San Jose, Calif). CD45-allophyco-

cyanain and CD8-PE were from Miltenyi Biotec. MEM-E/02 (anti–HLA-E

specific) was from Exbio (Praha, Czech Republic), anti-NKG2C was from

R&D Systems (Minneapolis, Minn), and W6/32 (anti-HLA class I) was

from Sigma-Aldrich.

Flow cytometryFlow cytofluorimetric analyses were performed on a FACSCalibur

cytofluorimeter with CellQuest software (BD Biosciences). Surface immu-

nofluorescence staining was conducted according to standard procedures by

using the appropriate labeled or unlabeled mAbs. In the latter case, staining

was followed by an FITC-conjugated polyclonal rabbit anti-mouse F(ab9)2

from Dako Cytomation (Glostrup, Denmark).

The expression of HLA molecules in patient and control keratinocytes was

performed by excluding dead cells from the analysis by means of propidium

iodide (0.5 mg/mL) staining in cell suspensions obtained from affected skin

biopsy specimens and from surgical operations of control donors. HLA

expression was determined in live CD452 cells with specific mAbs, and back-

ground mean fluorescence intensity derived from staining with control IgG1

mAb was subtracted from the mean fluorescence intensity determined for

the experimental mAb (W6/32 or 3D12).

FasL and IFN-g quantificationThe concentration of FasL and IFN-g in sera and blister fluids from patients

with SJS and TEN and healthy donors was measured by means of flow

cytometry with human FasL and IFN-g cytometric bead array Flex Set Kits

(BD Biosciences), respectively, and according to the manufacturer’s

instructions.

Keratinocyte cell cultureCell suspensions were obtained by means of 2 steps of 20 minutes’

digestion with 0.1% Trypsin and 5 mmol/L EDTA of punch biopsy specimens

from acute skin eruptions or biopsy specimens from healthy donors. Cultures

of primary human keratinocytes were generated by seeding these cell

suspensions on irradiated feeder layers of 3T3 fibroblasts. The keratinocyte

growth medium was Dulbecco modified Eagle medium supplemented with

10% FCS, glutamine, hydrocortisone (0.4 mg/mL), insulin (5 mg/mL),

triiodothyronine (2 nmol/L), epidermal growth factor (10 ng/mL) and cholera

toxin (0.1 nmol/L). Epidermal growth factor was not included in the medium

at seeding. Cultures were fed with keratinocyte medium 3 times per week. Ke-

ratinocytes were routinely subcultured onto fresh feeder cells after reaching

80% confluence after the selective removal of the feeders with EDTA

(0.02% in PBS) and trypsinization.

IFN-g treatment in keratinocytesThe HaCaT cell line and keratinocytes from skin biopsy specimens from

healthy donors were treated with 2.5 or 10 ng/mL rhIFN-g (Peprotech),

respectively, for 48 hours.

ImmunohistochemistryParaffin sections (5 mm thick) were mounted on pretreated slides,

deparaffinized with xylol, rehydrated through a graded series of ethanol,

and rinsed in distilled water. The endogen peroxidase activity was blocked

with 3% H2O2 for 20 minutes, and tissue sections were then boiled with citrate

buffer (pH 6.0). Nonspecific binding was prevented by applying 1% BSA

(Sigma-Aldrich), human IgG (50 mg/mL), and 0.3% Triton X-100 for 3 hours

before staining with 10 mg/mL of the anti–HLA-E–specific mAb MEM-E/02

for 72 hours at 48C. The sections were incubated with horseradish peroxidase–

conjugated anti-mouse antibody (Chemicon International) for 1 hour at room

temperature. Immunostaining was visualized with the DAB Substrate Kit (In-

vitrogen, Carlsbad, Calif), and tissues were counterstained with hematoxylin.

Statistical analysisCell-surface receptor data from patients with MPE and SJS/TEN; CD94/

NKG2C expression data in CD31, CD31CD81, and/or CD3– peripheral blood

lymphocytes from healthy donors’ and patients’ samples; HLA-E expression

data from keratinocytes from healthy donors and patients; and FasL and IFN-g

concentrations in sera from patients and healthy donors were analyzed for sta-

tistical significance by using the Mann-Whitney U test for unpaired samples.

The Wilcoxon matched paired test was used to compare NKG2A expression,

NKG2C expression, or both in paired samples from patients and healthy do-

nors; cytotoxic activity of PBMCs and BFCs against 721.221 and 221.AEH

cells; and FasL and IFN-g concentration in sera and blister fluids from pa-

tients. In all cases data are expressed as means and SEMs. Statistical analysis

was performed by using the GraphPad Prism software package (GraphPad

Software, Inc, San Diego, Calif).

FIG E1. Flow cytometric analysis of cell-surface receptor expression on T lymphocytes from peripheral

blood and blister fluids from patients with SJS/TEN. Panels show frequencies of CD81, CD941, CD561,

KIR2DL2/L3/S21, HLA-DR1, and CD251 cells in CD31 blister fluid and peripheral blood lymphocytes from

acute and resolution samples in a representative patient with TEN.

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FIG E2. FasL and IFN-g levels in sera and blister fluids from patients with

SJS/TEN. Graphs show picograms of FasL and IFN-g per milliliter of serum

or blister fluid from patients’ acute samples and healthy donors (HD). Hor-

izontal bars correspond to mean values. *P value, Wilcoxon matched pair

test; #P value, Mann-Whitney U test.

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FIG E3. HLA -E–specific CD561CD81 T-cell clones are cytotoxic against IFN-g–stimulated keratinocytes ex-

pressing HLA-E. A, HLA-I and HLA-E expression in primary keratinocytes and HaCaT cells stimulated or not

with IFN-g. Staining with control IgG1 and specific mAbs is shown. B and C, Cytotoxicity mediated by

CD561CD81 T-cell clones against HaCaT keratinocytes pretreated or not with IFN-g. The 721.221 cell line

and its HLA-E transfectant, 221.AEH, were used as negative and positive controls, respectively. Fig E3, B,

Percentages of lysis from 3 representative clones of 5 tested. Fig E3, C, Lysis in the presence of anti HLA-

E and anti-HLA-I mAbs.

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FIG E4. Flow cytometric analysis of receptor expression in PBMCs. A, Percentages of CD941, CD94/

NKG2A1, and CD94/NKG2C1 cells in CD31 and CD32 peripheral blood lymphocytes from patients with

SJS/TEN (acute samples) and healthy donors (HD). B, Percentages of CD94/NKG2C1 cells (means 6

SEMs) in CD31CD81 and CD32 peripheral blood lymphocytes from acute samples in patients with SJS/

TEN and MPE . *P value, Wilcoxon matched pair test; #P value, Mann-Whitney U test.

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FIG E5. CD94/NKG2C expression on peripheral blood lymphocytes and blasts in acute and resolution

samples from patients with drug-induced cutaneous reactions. A, CD3 and CD8 expression and forward

scatter (FSC) analysis in total PBMCs from a representative patient. Red- and blue-colored populations cor-

respond to lymphocytes and blasts, respectively. B, Panels show percentages of CD31CD81, CD94/NKG2C1,

and CD32CD94/NKG2C1 cells in peripheral blood lymphocytes (red gates) or blasts (blue gates) in 3

patients.

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TABLE E1. Patients with bullous reactions

Patient ID Sex Age (y) Drug or drugs involved

TEN 1 M 37 Ibuprofen

TEN 2 F 28 Erythromycin,

acetaminophen

TEN 3 M 55 allopurinol

SJS 1 F 27 Spiramycin,

metronidazole,

ibuprofen

SJS 2 F 39 Amoxicillin, clavulanic

acid, ibuprofen

SJS 3 M 67 Phenytoin

SJS 4 F 82 Undetermined

SJS 5 F 45 Undetermined

SJS 6 F 64 Undetermined

SJS 7 F 74 Amoxicillin, clavulanic

acid

SJS 8 F 38 Ibuprofen

SJS 9 F 17 Anticonvulsant

SJS 10 M 8 Anticonvulsant

SJS 11 F 73 Anticonvulsant

SJS 12 M 62 Isoniazid

SJS 13 M 2 Cefotaxime, vancomycin,

metronidazole

SJS 14 M 2 Cefotaxime, clindamycin

SJS 15 F 54 Doxorubicin

F, Female; M, male.

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TABLE E2. Patients with drug hypersensitivity reactions included in the study

Solid squares indicate figures in which samples from each patient were included.

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