Allele-Specific Cytokine Responses at theHLA-C Locus: Implications for PsoriasisChristian Hundhausen1,7, Anna Bertoni2,7, Rose K. Mak1, Elisabetta Botti1,3, Paola Di Meglio1, Alex Clop2,Ute Laggner1, Sergio Chimenti3, Adrian C. Hayday4,5, Jonathan N. Barker1, Richard C. Trembath2,6,Francesca Capon2 and Frank O. Nestle1

Psoriasis is an inflammatory skin disorder that is inherited as a complex trait. Genetic studies have repeatedlyhighlighted HLA-C as the major determinant for psoriasis susceptibility, with the Cw*0602 allele conferringsignificant disease risk in a wide range of populations. Despite the potential importance of HLA-C variation inpsoriasis, either via an effect on peptide presentation or immuno-inhibitory activity, allele-specific expressionpatterns have not been investigated. Here, we used reporter assays to characterize two regulatory variants,which virtually abolished the response to tumor necrosis factor (TNF)-a (rs2524094) and IFN-g (rs10657191) inHLA-Cw*0602 and a cluster of related alleles. We validated these findings through the analysis of HLA-Cw*0602expression in primary keratinocytes treated with TNF-a and IFN-g. Finally, we showed that HLA-Cw*0602transcripts are not increased in psoriatic skin lesions, despite highly elevated TNF-a levels. Thus, our findingsdemonstrate the presence of allele-specific differences in HLA-C expression and indicate that HLA-Cw*0602 isunresponsive to upregulation by key proinflammatory cytokines in psoriasis. These data pave the way forfunctional studies into the pathogenic role of the major psoriasis susceptibility allele.

Journal of Investigative Dermatology (2012) 132, 635–641; doi:10.1038/jid.2011.378; published online 24 November 2011

INTRODUCTIONPsoriasis is a chronic, inflammatory skin disorder, affectingapproximately 2% of the Caucasian population. The disease ischaracterized by keratinocyte hyperproliferation and altereddifferentiation, in the presence of an inflammatory skin infiltrate,typically consisting of dendritic cells, macrophages, T cells, andneutrophils (Nestle et al., 2009). Psoriasis has a strong geneticcomponent and is widely regarded as a multifactorial disorder,resulting from gene–gene and gene–environment interactions(Griffiths and Barker, 2007; Liu et al., 2007).

A highly significant association between psoriasis and theHLA-Cw6 antigen has long been established (Henseler and

Christophers, 1985; Mallon et al., 1999). In agreement withthese historical observations, genome-wide linkage scanshave repeatedly identified a primary disease susceptibilitylocus (PSORS1), lying within the class I region of the majorhistocompatibility complex, on chromosome 6p21.3 (Nairet al., 1997; Trembath et al., 1997). Refinement studies, deepsequencing experiments, and recent genome-wide associa-tion data all point to HLA-C as the most likely PSORS1gene (Veal et al., 2002; Nair et al., 2006; Liu et al., 2008;Strange et al., 2010). However, the extended linkagedisequilibrium that characterizes the major histocompatibilitycomplex region has complicated the analysis of thePSORS1 locus and has so far prevented the identification ofthe casual susceptibility variant. At the same time, theanalysis of PSORS1 sequences associated with differentHLA-C alleles has demonstrated that the single-nucleotidepolymorphisms (SNPs) that are unique to HLA-Cw*0602haplotypes lie exclusively in noncoding regions (Nair et al.,2006).

In this context, the hypothesis driving this study is that thePSORS1 disease susceptibility allele may lie within aregulatory region influencing HLA-C expression. To explorethis pathogenic model, we investigated the differentialregulation of three HLA-C alleles, using reporter assays, incombination with patient-based expression studies. Thisintegrated approach demonstrated the previously unreportedexistence of allele-specific HLA-C expression patterns andspecifically identified two regulatory variants affecting theresponse to IFN-g and tumor necrosis factor (TNF)-a.

& 2012 The Society for Investigative Dermatology www.jidonline.org 635

ORIGINAL ARTICLE

Received 23 May 2011; revised 19 August 2011; accepted 5 October 2011;published online 24 November 2011

1St John’s Institute of Dermatology, King’s College London, Guy’s Hospital,London, UK; 2Department of Medical and Molecular Genetics, King’s CollegeLondon, Guy’s Hospital, London, UK; 3Department of Dermatology,University of Rome Tor Vergata, Rome, Italy; 4Division of Immunology,Infection and Inflammatory Disease, King’s College London, Guy’s Hospital,London, UK; 5London Research Institute, Cancer Research UK, London, UKand 6Barts and the London School of Medicine and Dentistry, Queen MaryUniversity of London, London, UK

Correspondence: Frank O. Nestle, King’s College London, St John’s Instituteof Dermatology, Guy’s Hospital, London, UK. E-mail: frank.nestle@kcl.ac.ukor Francesca Capon, King’s College London, Department of Medical andMolecular Genetics, Guy’s Hospital, London, UK.E-mail: francesca.capon@kcl.ac.uk

7These authors contributed equally to this work

Abbreviations: PSORS, psoriasis susceptibility locus; TNF, tumor necrosisfactor

RESULTSTwo sequence variants in the HLA-C promoter causenonresponsiveness to TNF-a and IFNs

To assess the presence of HLA-Cw*0602-specific geneexpression patterns, we measured the reporter activity drivenby the HLA-Cw*0602, -Cw*0702, and -Cw*0304 promoters.We examined a 426-bp fragment corresponding to thegene minimal promoter (Johnson, 2003) and found that theHLA-Cw*0602 reporter construct was significantly less activethan its HLA-Cw*0702 counterpart (Figure 1a). Importantly,the HLA-C promoter contains an experimentally definedenhancer kB element (Johnson and Pober, 1994), whichdisplays a G to A substitution (SNP rs2524094, seeSupplementary Figure S1 online) in HLA-Cw*0602 and in acluster of related alleles. To investigate whether this poly-morphism was responsible for the differential activity of theHLA-Cw*0602 and -Cw*0702 promoters, we repeatedthe reporter assays following site-directed mutagenesis ofthe HLA-Cw*0702 enhancer kB. These experiments revealeda significant decrease in reporter activity for the mutagenizedconstruct (Figure 1b), thus confirming the functional impactof the rs2524094 polymorphism.

To investigate whether SNP rs2524094 also affectsinducible promoter activity, we next carried out reporterassays in cells that had been stimulated with TNF-a, IL-17A,and IL-22, three inflammatory cytokines that signal throughthe NF-kB pathway and have a key role in the pathogenesisof psoriasis (Nickoloff et al., 2007). Surprisingly, none of theexamined HLA-C alleles responded to IL-17A or IL-22treatment (Figure 1f). Conversely, the HLA-Cw*0702 promo-ter displayed significant TNF-a–induced luciferase activity,in keeping with the role of this cytokine as an inducer ofmajor histocompatibility complex gene expression (Johnson,2003). At the same time, HLA-Cw*0602 showed a weak andnonsignificant TNF-a response (Figure 1c). The analysis ofmutagenized constructs demonstrated that the introduction ofa wild-type rs2524094 allele in the HLA-Cw*0602 promoterrestored TNF-a responsiveness (Figure 1e). Conversely,disruption of the HLA-Cw*0702 enhancer kB resulted in amore modest TNF-a response (Figure 1d). Thus, the TNF-a–induced activity of HLA-Cw*0702 is dependent on theintegrity of the enhancer kB element.

The HLA-C minimal promoter also contains an IFNresponse stimulated element (ISRE), similar to the onemediating IFN induction of the HLA-A and HLA-B genes(Johnson, 2003). It is noteworthy that the HLA-C ISRE has adifferent base composition in HLA-Cw*0602 and HLA-Cw*0702, due to the occurrence of a 3 bp deletion in thelatter allele (-166_-163delTCT, rs10657191; SupplementaryFigure S1 online). Reporter assays showed that HLA-Cw*0702, but not HLA-Cw*0602, could respond to IFNstimulation. The analysis of a mutagenized construct con-firmed that the ISRE deletion polymorphism was responsiblefor this difference (Figure 1g and h).

Harboring the same kB and ISRE elements as HLA-Cw*0602, HLA-Cw*0304 served as control and showedsimilar reporter activity to HLA-Cw*0602 throughout allexperiments (Figure 1a, c, f, g and h).

Taken together, these results demonstrate that thers2524094 and the rs10657191 promoter variants affectHLA-C cytokine responses, with the A and insertion allelesdetermining nonresponsiveness to TNF-a and IFNs.

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Figure 1. HLA-C promoter variants determine nonresponsiveness of

HLA-Cw*0602 to TNF-a and IFNs. HeLa (a–f) or HEK293T (g–h) cells

were transfected with a luciferase gene under the control of wild-type

and mutagenized HLA-C promoters, along with a control reporter gene

encoding for b-galactosidase. (c–h) Transfected cells were treated with the

indicated cytokines for 24 hours and a dual-light reporter assay was

performed, in order to measure luciferase (luc) and b-galactosidase

(gal) activities. The results are expressed as the mean±SEM of luciferase/

b-galactosidase ratios. The Cw*06 luciferase/b-galactosidase ratio was

set as baseline to which all other measurements were normalized. Each

experiment was performed at least three times in triplicate. Statistical

significance was calculated using one-way analysis of variance (ANOVA,

a), t-test (b), or two-way ANOVA (c–h). ***Po0.001, **Po0.01, *Po0.05.

kBmut refers to the site-directed mutagenesis performed in the enhancer

kB of the -Cw*0602 and -Cw*0702 alleles. del-ISRE refers to the introduction

of a 3 bp deletion in the -Cw*0602 ISRE. ISRE, IFN response stimulated

element.

636 Journal of Investigative Dermatology (2012), Volume 132

C Hundhausen et al.HLA-Cw*0602 in Psoriasis

TNF-a treatment of primary keratinocytes fails to upregulateHLA-Cw*0602

Having established that the HLA-Cw*0602 promoter displaysreduced cytokine responses in reporter assays, we proceededto investigate HLA-C expression in cultured primary kerati-nocytes. As the presence of inflammatory cytokines inpsoriatic skin would have confounded the interpretation ofour results, we carried out these experiments in cells isolatedfrom healthy donor skin samples. In keeping with the resultsobtained in reporter assays, neither IL-17A nor IL-22 had asignificant effect on HLA-C expression, regardless of theHLA-C genotype (Figure 2a, b and d). In both HLA-Cw*0602-positive and -negative keratinocytes, IFN-g was the onlysingle cytokine that profoundly upregulated HLA-C

(Figure 2a and b). The strong synergistic effect of TNF-a incombination with IFN-g as observed for donor N1 (Figure 2a)was significantly reduced in HLA-Cw*0602-positive kerati-nocytes (donor N2, Figure 2a). Following the validationof antibody specificity (Supplementary Figure S2a online),western blotting for HLA-C was performed, confirming themRNA data at the protein level (Figure 2b). To analyzecytokine-induced HLA-C cell surface expression, immuno-fluorescence staining was also performed on keratinocytesfrom donor N1. The results confirmed IFN-g as the strongestmodulator of HLA-C expression (Figure 2c).

To specifically investigate the regulation of HLA-Cw*0602transcripts, we next designed allele-specific, exon-spanningprimers (Supplementary Figure S2b online), which we used in

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Figure 2. Cytokine-induced HLA-C expression in primary keratinocytes. (a) Primary keratinocytes derived from healthy donors N1 and N2 were treated

with tumor necrosis factor (TNF)-a (10 ng ml�1), IFN-g (500 U ml�1), TNF-a (10 ng ml�1) þ IFN-g (500 U ml�1), IL-17A (10 ng ml�1), and IL-22 (10 ng ml�1)

for 24 hours or left untreated. For each condition, cells were split for RNA and protein isolation. HLA-C transcript levels were measured by real-time PCR

and normalized to the mean of cyclophylin A and Rplpo (large ribosomal protein) expression. HLA-C expression levels are plotted as fold changes

compared with the untreated control. Significance values were calculated using one-way analysis of variance. ***Po0.001, **Po0.01, *Po0.05. (b) 1% Triton

lysates of keratinocytes derived from donors N1 and N2 were analyzed for HLA-C protein expression by western blotting. (c) Immunofluorescence staining

of HLA-C. Cytokine-treated keratinocytes from donor N1 were deposited on glass slides by Cytospin and stained for HLA-C protein (red). Nuclei were

counterstained with 4,6-diamidino-2-phenylindole (blue). Bar¼ 50mm. (d) Total HLA-C and HLA-Cw*0602 mRNA levels were determined in primary

keratinocytes derived from three HLA-Cw*0602 heterozygous donors. Cytokine stimulation, RNA extraction, and complementary DNA synthesis were

carried out as described in a. Relative mRNA levels were expressed as fold change compared with the untreated control. White bars indicate total HLA-C

and green bars HLA-Cw*0602 expression. Significance values refer to differences in fold induction.

www.jidonline.org 637

C Hundhausen et al.HLA-Cw*0602 in Psoriasis

real-time PCR measurements of gene expression. The resultsof these experiments were again consistent with thoseobtained in reporter assays. Total HLA-C and HLA-Cw*0602 mRNA levels were both significantly upregulatedin response to IFN-g; however, HLA-Cw*0602 expressionwas markedly less enhanced (Figure 2d). Moreover, TNF-atreatment had no effect on HLA-Cw*0602 transcript levels,and total HLA-C expression increased only in the cellsderived from donor N3 (Figure 2d,), whose non-risk allele(Cw*17) carries a canonical enhancer kB sequence (Supple-mentary Figure S1 online).

Thus, the analysis of endogenous HLA-Cw*0602 expres-sion in human keratinocytes confirmed the nonresponsive-ness to TNF-a, which we had observed in our reporter assays.

HLA-Cw*0602 expression levels do not differ significantly innormal, peri-lesional, and lesional skin

In the final phase of this study, we investigated HLA-Cexpression in whole tissue by analyzing total RNA isolatedfrom normal skin (N) (n¼ 31) and from matched pairs of peri-lesional and lesional (L) psoriasis skin biopsies (n¼26). Wefound that there was variation in non-risk HLA-C transcriptlevels (Figures 3a), potentially reflecting the presence ofvarious combinations of regulatory elements in the differentnon-risk alleles (Supplementary Table S1 online and Supple-mentary Figures S1 and S3 online). In the geneticallyhomogenous HLA-Cw*0602 chromosome population, nosignificant upregulation of HLA-Cw*0602 gene expressionwas detected between normal, peri-lesional, and lesionalsamples (Figure 3b), despite the presence of elevated TNF-aand IFN-g levels in psoriatic skin (Supplementary Figure S4online). The amount of total HLA-C protein found in the

epidermis was variable, but no evidence of upregulation wasobserved in lesional (highlighted with an L in Figure 3c)versus non-lesional (peri-lesional (PL)) samples, obtainedfrom HLA-Cw*0602 heterozygous individuals (Figure 3c).

Our data also show that there is no correlation betweenHLA-C expression and psoriasis area and severity index score(Supplementary Figure S5 online) and that transcript levelsare not affected by the use of topical or systemic therapeuticagents (Supplementary Figure S6 online). Thus, our results areunlikely to be confounded by individual variation in diseaseseverity or treatment.

Taken together, these findings are in agreement with thenotion of HLA-Cw*0602 nonresponsiveness to TNF-a, as theyshow that HLA-Cw*0602 gene expression remains largelyunchanged in psoriatic skin, despite the presence of elevatedcytokine levels.

DISCUSSIONThis study explores allele-specific patterns of HLA-C regula-tion, using a combination of reductionist promoter assays andstudies in human tissue samples.

Our work was driven by the results of serologic studies,which have long established an unequivocal associationbetween HLA-Cw6 and psoriasis (Henseler and Christophers,1985; Mallon et al., 1999) and those of recent genome-wideassociation scans, which have highlighted HLA-C as the mostlikely PSORS1 candidate gene (Ellinghaus et al., 2010;Strange et al., 2010; Stuart et al., 2010; Sun et al., 2010).Because of its function as innate and adaptive immuno-regulator, an involvement of HLA-C in psoriasis pathogenesisis conceivable. HLA-Cw6 in particular has peptide-presentingcapacity (Falk et al., 1993; Dionne et al., 2004) and can

HLA-C (non-risk) HLA-Cw*0602

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Figure 3. HLA-C expression levels in normal and psoriatic skin. (a, b) HLA-C and HLA-Cw*0602 mRNA expression was measured in normal skin (N)

and in matched pairs of peri-lesional (PL) and lesional (L) psoriatic skin. (a) HLA-C expression in HLA-Cw*0602-negative samples of N (n¼21), PL (n¼10),

and L (n¼ 10) skin. (b) HLA-Cw*0602 expression in -Cw*0602 þ /� samples of N (n¼ 10), PL (n¼ 16), and L (n¼ 16) skin. Real-time data are presented

as individual data points normalized to expression of the internal controls GAPDH (a) and b-actin (b). One-way analysis of variance statistical test was performed

for a and b yielding P-values 40.05. (c) Western blotting for HLA-C protein in the epidermis of normal and psoriatic skin. Green font indicates samples

heterozygous for HLA-Cw*0602, black font HLA-Cw*0602-negative samples.

638 Journal of Investigative Dermatology (2012), Volume 132

C Hundhausen et al.HLA-Cw*0602 in Psoriasis

trigger CD8þ T-cell–specific responses (Dionne et al., 2004;Johnston et al., 2004).

Although total HLA-C expression has previously beeninvestigated in psoriasis (Zhou et al., 2003; Carlen et al.,2005, 2007; Nair et al., 2009), allele-specific patterns havenever been examined. In this context, our study providesimportant insights into the regulation and expression of therisk allele, HLA-Cw*0602, in normal, peri-lesional, andlesional psoriatic skin.

We have used a combination of reporter and real-timePCR assays to characterize a promoter variant (rs2524094)mediating reduced responsiveness to TNF-a. We also investi-gated an insertion/deletion polymorphism (rs10657191)and found that it determined reduced sensitivity to IFNsin reporter assays. However, this effect was not replicatedin real-time PCR experiments, where differential IFNresponses were observed in alleles sharing the same ISREelement (Figure 2d). Thus, we suggest that other regulatoryelements outside of the HLA-C minimal promoter might beof importance for the IFN response. We further argue that theTh17 association with psoriasis (Di Cesare et al., 2009) is notrelated to an effect on HLA-C transcript levels, as both IL-17Aand IL-22 failed to alter HLA-C promoter activity orendogenous expression. Our observation that HLA-Cw*0602 transcript levels do not differ significantly betweennormal and psoriatic skin is in keeping with the abovefindings, and further support the notion of HLA-Cw*0602reduced responsiveness to proinflammatory cytokines.

It is worth pointing out that the rs2524094 variant is notunique to HLA-Cw*0602, and thus other HLA-C alleles arelikely to show reduced responsiveness to TNF-a. In fact, ourreporter assays show that this is the case for HLA-Cw*0304,which harbors the same enhancer kB element as HLA-Cw*0602. At the same time, reduced responsiveness to TNF-a is likely to have a different impact on risk and non-riskHLA-C alleles, as they are likely to have differential affinitiesfor self-peptides and to interact with different bindingpartners. For instance, the HLA-Cw6 (but not the HLA-Cw3)protein has been shown to interact with KIR2DL1 inhibitoryreceptor, which regulates the activity of natural killer andT-cell subsets (Parham, 2005). It is noteworthy that associa-tions between psoriasis and KIR receptor polymorphismshave been reported in the past (Luszczek et al., 2004), with anumber of studies suggesting pathogenic and protective rolesfor specific HLA-C/KIR genotype combinations (Nelson et al.,2004; Holm et al., 2005). Further functional experiments willnow be required to explore the impact of TNF-a nonrespon-siveness on HLA-C/KIR interactions and self-peptide pre-sentation. Such studies hold the promise to elucidate themolecular mechanisms underlying the contribution of HLA-Cw*0602 to psoriasis susceptibility.

MATERIALS AND METHODSStudy populationBlood samples and skin biopsies were obtained from 26 patients

with plaque psoriasis (Supplementary Table S1 online). Control

skin tissue was obtained from 31 healthy volunteers undergoing

breast or abdominal reduction surgery. Our study was conducted in

accordance with the Declaration of Helsinki Principles, with

informed consent obtained from each volunteer and ethical approval

granted by the institutional review board of Guy’s Hospital.

Cell culture and cytokine stimulation assays

HeLa and HEK293T cell lines were cultured in DMEM medium

(Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum

(Sigma-Aldrich, St Louis, MO) and 1% penicillin/streptomycin (PAA,

Yeovil, Somerset, UK). LCL721.221-Cw*0602, LCL721.221-mock,

and HLA class I K562 transfectants were kindly provided by Dr

Matthias Marget (Institute of Immunology, University Medical Center

Schleswig-Holstein, Germany), and Dr Eric Champagne (INSERM

Toulouse, France), respectively; both LCL721.221 and K562 cells

were cultured in RPMI 1640 medium (Invitrogen) supplemented with

10% fetal bovine serum and 1% penicillin/streptomycin.

Primary keratinocyte cell cultures were established using the

procedure described by Mee et al. (2007). Keratinocytes were

cultured for 2–4 passages in KGM-2 medium, in the presence of

growth supplements (KGM-2 Bullet Kit CC-3107, Lonza, Slough,

UK). For cytokine treatment, cells were stimulated for 24 hours with

either 10 ng ml�1 TNF-a, 500 U ml�1 IFN-g (PeproTech, London,

UK), 10 ng ml�1 IL-17A, or IL-22 (R&D Systems, Abingdon, UK).

Plasmids

Three BAC clones carrying HLA-C, but not HLA-A or HLA-B, were

used as templates for the specific amplification of the HLA-C

promoter. In particular, the BACs containing the Cw*0602 and

Cw*0702 promoters were selected from a library, which had been

previously generated from the leukocytes of a heterozygous psoriatic

patient, using the procedure described by Osoegawa et al. (2001). A

third BAC, which carries the HLA-Cw*0304 promoter, was kindly

provided by Dr Lucy Matthews (Wellcome Trust Sanger Institute,

Cambridge, UK). Allele-specific oligonucleotides tagged with XhoI

and HindIII (New England Biolabs, Hitchin, UK) restriction sites

were used to clone the minimal HLA-C promoter (Johnson, 2003)

into the pGL3-basic vector (Promega, Southampton, UK). Primer

sequences were HLA-C.F 50-AATCGCTCGAGAGGGACGGGGAT

TCCAGGAG-30, HLA-Cw*0602/*0304.R 50-AGGCAAGCTTCTCGG

CGTCTGGGGAGA-30, HLA-Cw*0702.R 50-AGGCAAGCTTCTC

GGCCTCTGGGGAGA-30.

Mutagenized constructs were generated using the QuikChange

site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to

manufacturer’s instructions. The presence of the correct insert was

verified by direct sequencing of all wild-type and mutant constructs.

Transient transfection and dual-light reporter assayHeLa or HEK293T cells were transfected with 1mg pGL3-basic

vector and 0.45 mg pJ7lacZ vector (kindly provided by Dr Talat

Nasim, King’s College London, UK), using the FuGene HD trans-

fection reagent (Roche, Mannheim, Germany). Cells were harvested

24 hours post transfection, and reporter gene expression was

measured with the Dual-Light luminescent reporter gene assay

(Applied Biosystems, Bedford, UK), according to the manufacturer’s

instructions.

RNA isolation and real-time PCR

Total RNA was isolated from cell lines and primary human keratino-

cytes using the RNeasy Plus Mini Kit (Qiagen, Crawley, UK) or TRI

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C Hundhausen et al.HLA-Cw*0602 in Psoriasis

REAGENT (Sigma-Aldrich), according to the manufacturer’s protocol.

A measure of 500–1,000 ng total RNA was reverse transcribed into

complementary DNA, using SuperScript II Reverse Transcriptase

(Invitrogen) according to the manufacturers’ instructions.

Total HLA-C and HLA-Cw*0602 mRNA levels were assessed

by quantitative PCR (gene expression assay Hs03044135_m1) and

SYBR Green–based PCR, respectively. Primers for specific detection

of the HLA-Cw*0602 transcript were as follows: forward 50-TACTA

CAACCAGAGCGAGGA-30 and reverse 50-GGTCGCAGCCATACAT

CCA-30. PPIA, GAPDH b-actin transcripts were used as endogenous

controls. Samples were amplified on a Applied Biosystems 7900HT

Sequence Detection System, and real-time data were presented

either as individual data points normalized to the internal control

(2�DCt) or expressed as fold change using the comparative Ct (2�DDCt)

method (Schmittgen and Livak, 2008).

HLA-C locus typing

Genomic DNA was extracted from peripheral blood or skin, using

the DNeasy Blood & Tissue Kit (Qiagen). Medium resolution HLA-C

typing was performed using PCR with sequence-specific primers

(Bunce et al., 1995).

Western blottingCell and tissue lysates were analyzed by western blotting, using

1mg ml�1 anti-HLA-C goat polyclonal antibody Q-18 (Santa Cruz

Biotechnology, CA) together with 1mg ml�1 anti-HSP90 rabbit mAb

H-114 (Santa Cruz Biotechnology) or anti-b-actin mouse mAb AC-15

(1:2,000, Sigma-Aldrich) as endogenous controls.

Cytospin and immunofluorescence

Cytospin (Cytospin 3 Cytocentrifuge, Thermo Fisher Scientific,

Loughborough, UK) was used to deposit monolayers of 2� 105

primary human keratinocytes on glass slides. Cells were fixed

with acetone at �20 1C and incubated with HLA-C (Q18) goat

polyclonal IgG antibody (1:50 dilution) for 1 hour at room

temperature. Following incubation with donkey anti-goat Alexa

555 IgG antibody (1:200 dilution), cells were mounted with Prolong

Gold antifade reagent supplemented with 4,6-diamidino-2-pheny-

lindole (Invitrogen). Pictures were obtained using a fluorescent

microscope (Zeiss Axiophot Microscope, Carl Zeiss Jena GmbH,

Jena, Germany) with a camera (Nikon Digital Sight Camera, Nikon,

Tokyo, Japan).

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSWe thank the psoriasis patients and healthy volunteers who took part in thisstudy. We also thank Isabella Tosi, Gayathri Perera, and Robert Vaughan fortissue collection and HLA-C typing. We acknowledge support from theDepartment of Health via the National Institute for Health Research (NIHR)comprehensive Biomedical Research Centre award to Guy’s & St Thomas’NHS Foundation Trust in partnership with King’s College London and King’sCollege Hospital NHS Foundation Trust. This work was funded by the NationalInstitutes of Health (grant RO1 AR040065 to FON), The Wellcome Trust(Programme grant GR078173MA, to FON), the Medical Research Council(Programme grant G0601387 to RCT, FON, ACH, JNB), the British SkinFoundation (grant 1006 to FC), Dunhill Medical Trust, a Medical ResearchCouncil Clinical Research Fellowship (to RKM, grant GO700553), and aGeneration Trust PhD studentship (AB).

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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