Journal of Neuroimmunology xxx (2014) xxx–xxx

JNI-475943; No of Pages 6

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Oligoclonal band phenotypes inMS differ in their HLA class II association,while specific KIR ligands at HLA class I show association toMS in general

Marte W. Gustavsen a,b,⁎, Marte K. Viken c,d,e, Elisabeth G. Celius a, Tone Berge a, Inger-Lise Mero c,Pål Berg-Hansen a,b, Jan H. Aarseth f, Kjell-Morten Myhr f,g, Helle B. Søndergaard h, Finn Sellebjerg h,Annette B. Oturai h, Jan Hillert i, Lars Alfredsson j, Tomas Olsson k, Ingrid Kockum k,Benedicte A. Lie c,d,e, Hanne F. Harbo a,b

a Department of Neurology, Oslo University Hospital, Ullevål, Oslo, Norwayb Institute of Clinical Medicine, University of Oslo, Oslo, Norwayc Department of Medical Genetics, University of Oslo and Oslo University Hospital, Oslo, Norwayd Department of Immunology, Oslo University Hospital and University of Oslo, Oslo, Norwaye K.G. Jebsen Inflammation Research Centre, University of Oslo, Oslo, Norwayf The Norwegian Multiple Sclerosis Registry and Biobank, Department of Neurology, Haukeland University Hospital, Bergen, Norwayg KG Jebsen Centre for MS-research, Department of Clinical Medicine, University of Bergen, Bergen, Norwayh The Danish Multiple Sclerosis Center, Department of Neurology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmarki Department of Clinical Neuroscience, Karolinska Institutet, The Multiple Sclerosis Research Group, Center for Molecular Medicine, L8:00, Stockholm, Swedenj Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Swedenk Department of Clinical Neuroscience, Karolinska Institutet, The Neuroimmunology Research Group, Stockholm, Sweden

⁎ Corresponding author at: Department of Neurology, Oand University of Oslo, N-0407 Oslo, Norway. Tel.: +47 2

E-mail address: m.w.gustavsen@medisin.uio.no (M.W

http://dx.doi.org/10.1016/j.jneuroim.2014.06.0240165-5728/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Gustavsen, M.W.,ligands at HLA class I show associa..., J. Neur

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 April 2014Received in revised form 25 June 2014Accepted 27 June 2014Available online xxxx

Keywords:Multiple sclerosisOligoclonal bandsHLAKiller cell immunoglobulin-like receptors

Multiple sclerosis (MS) patients have been reported to have different HLA class II allele profiles depending onoligoclonal bands (OCBs) in the cerebrospinal fluid, but HLA class I alleles and killer cell immunoglobulin-like re-ceptor (KIR) ligands have not been studied.We investigated the association ofHLA alleles andKIR ligands accord-ing to OCB status inMS patients (n= 3876). Specific KIR ligands were associated with patients when comparedto controls (n = 3148), supporting a role for NK cells in MS pathogenesis. HLA class I alleles and KIR ligands didnot differ between OCB phenotypes, but HLA class II associations were convincingly replicated.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis (MS) is an inflammatory disease of the centralnervous system that leads to damage of myelin and degeneration ofaxons (Compston and Coles, 2008). In recent years, the knowledge ofgenetic and environmental risk factors has increased extensively, butthe cause of MS is still unknown. Human leukocyte antigen (HLA) lociwere shown to be associated with MS already in the 1970's (Jersildet al., 1972), and theHLA-DRB1*15:01 allele has consistently been iden-tified as the strongest genetic risk factor forMS (IMSGC&WTCCC2, 2011,Patsopoulos et al., 2013). Also, HLA class I alleles have been related toMS susceptibility (Link et al., 2010), with HLA-A*02:01 as the best

slo University Hospital Ullevål3016285; fax: +47 23027455.. Gustavsen).

et al., Oligoclonal band phenooimmunol. (2014), http://dx.d

established protective allele (Brynedal et al., 2007, Link et al., 2010,IMSGC&WTCCC2, 2011, Patsopoulos et al., 2013).

HLA class I molecules present peptides to CD8+ T cells, but are alsorecognized by other cells and their receptors, such as killer cellimmunoglobulin-like receptors (KIRs) (Trowsdale and Knight, 2013).KIRs are expressed mainly on natural killer (NK) cells (Vivier et al.,2008), but also on T cell subsets (Van Bergen et al., 2004). Although Tand B cells and the interplay between them are regarded as the mostcentral elements in the pathogenesis of MS (Weber and Hemmer,2010), there is increasing support for an influence also of innate im-mune cells, such as NK cells (Takahashi et al., 2004, Kaur et al., 2013),which have both cytotoxic and cytokine producing abilities. In additionto representing front-line defense against viruses and tumor cells, NKcells have important immunoregulatory functions and can regulateboth T cell and B cell responses (Vivier et al., 2008). KIR genes and KIRligands have been investigated in relation to MS susceptibility, age atonset, clinical course of MS and therapeutic response in MS

types in MS differ in their HLA class II association, while specific KIRoi.org/10.1016/j.jneuroim.2014.06.024

2 M.W. Gustavsen et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx

(Lorentzen et al., 2009, Fusco et al., 2010, Garcia-Leon et al., 2011,Martinez-Rodriguez et al., 2011, Jelcic et al., 2012, Bettencourt et al.,2014), but HLA class I alleles and their KIR binding motifs have notbeen studied in relation to oligoclonal band (OCB) status in MS.

OCBs in the cerebrospinal fluid (CSF), but not in correspondingserum, are detected in 90–95% of MS patients in Northern Europe(Link and Huang, 2006) and reflect an intrathecal IgG production of un-known etiology (Petzold, 2013). Several studies have shown that OCBnegative and positive MS patients differ both clinically and genetically.The OCB negative MS patients seem to have higher age at diseaseonset, lower disease severity scores and are more often men (Josephet al., 2009, Lechner-Scott et al., 2011, Mero et al., 2013). HLA-DRB1 al-leles have been investigated in relation to OCB status in several studies(Kikuchi et al., 2003, Imrell et al., 2006, Idiman et al., 2009, Wu et al.,2010, Romero-Pinel et al., 2011, Leone et al., 2013, Mero et al., 2013)with the main observations that the HLA-DRB1*04:04 allele confersrisk only for OCB negativeMS, whereas the HLA-DRB1*15:01 allele con-fers a stronger risk for OCB positiveMS thanOCB negativeMS. However,most previous studies have been modestly powered and the findingsneed confirmation. Also, no study has so far investigated a possible asso-ciation with HLA class I alleles to the OCB phenotypes.

On this background, we aimed to further investigate the genetic pro-file of the OCB phenotypes in MS, with emphasis on the distribution ofHLA class I alleles and relate this to their KIR binding motifs, both inthe different OCBphenotypes and in the total group ofMS patients com-pared to healthy controls.

2. Materials and methods

2.1. Patients and controls

Samples from 3876MS patients collected from Norway (n= 2143),Sweden (n= 1309) and Denmark (n= 424), and 3148 healthy controlsamples collected from Norway (n = 701) and Sweden (n = 2447)were included. All MS patients were diagnosed in accordance with theMcDonald criteria (Polman et al., 2011). The Norwegian controls wereblood donors registered in the Norwegian bonemarrow donor registry,and the Swedish controls were either recruited from the general popu-lation,matched toMS cases based on age, gender and residence or blooddonors from the Stockholm area. The clinical characteristics of the in-cluded patients and controls are presented in Table 1.

The study was approved by the regional ethical committees in eachcountry; The Regional Committee for Medical and Health ResearchEthics - South East and West (Norway), The Danish Ethical CommitteeReview Board for Copenhagen and Frederiksberg (Denmark) and TheRegional Ethical Review Board in Stockholm (Sweden).

Informed written consent was obtained for all Norwegian and Dan-ish samples included. The Swedish sampleswere collected in five differ-ent studies. Written approval was obtained from the patients andcontrols included in four of the studies, and informed oral approvalwas obtained for the fifth (including healthy blood donors and a

Table 1Characteristics of MS patients and controls.

MS patients, n = 3876 Controls, n = 3148

Gender female, n (%) 2775 (71.6) 2212 (70.3)RR-MS, n (%) 3449 (91.0) –

OCB in the CSF, n (%) 3450 (89.0) –

Median AAO 33.0 –

Median EDSS 3.00 –

Median MSSS 4.28 –

Abbreviations: RR: relapsing remitting, OCB: oligoclonal bands, CSF: cerebrospinal fluid,AAO: age at onset, EDSS: Expanded Disability Status Scale, MSSS: Multiple SclerosisSeverity Score.Missing values are not included in the calculations.

Please cite this article as: Gustavsen, M.W., et al., Oligoclonal band phenoligands at HLA class I show associa..., J. Neuroimmunol. (2014), http://dx.d

minority of the MS cases). Written approval was not mandatory at thetime of collection of these samples, but the use of these samples hasbeen approved by the Regional Ethical Review Board in Stockholm.

2.2. CSF analyses

Isoelectric focusing (IEF) with immunoblotting or agarose gel elec-trophoresis (AGE) was used to detect OCBs. IgG-index was calculatedfrom levels in CSF and serum (CSF/serum IgG:CSF/serum albumin). Apatient was classified as OCB negative in the absence of OCB detectionand as CSF positive if more than one OCB were detected in the CSFand/or in the presence of increased IgG index (N0.70).

2.3. HLA genotype data

Genotypes for four HLA loci (A, B, C andDRB1) at four digit resolutionwere imputed for 2574MS patients and 3148 healthy controls includedin the Immunochip project (IMSGC, 2013) using HLA*IMP2 (Diltheyet al., 2013). These imputed HLA genotypes from the Norwegian, Swed-ish andDanish individualswere pooled in a combined analysis as genet-ic homogeneity previously has been demonstrated for these cohorts(Mero et al., 2013). A fraction of these MS cases (n= 350) and controls(n=575) had been includedwithHLA-DRB1 genotype data at four digitresolution in a previous study (Mero et al., 2013), and 437MS cases and229 controls with HLA class I data had been included in another study(Lorentzen et al., 2009).

In addition to the imputed HLA-data, HLA-DRB1 genotypes at fourdigit resolution, obtained by a sequence based approach (Sayer et al.,2004), were available for 1302 additional Norwegian MS patients froma previous study (Mero et al., 2013). All samples with HLA-DRB1 datawere pooled in a combined analysis of 3876MS cases and 3148 controls.

As quality control, imputed data for theMS patients were comparedwith conventional sequencing data when this was available. The con-cordance rate between sequencing data and imputed data for HLA-Awas 97.7% (n = 463), for HLA-B 96.8% (n = 451) and for HLA-C 97.6%(n = 477) at two digit resolution. For HLA-DRB1, the concordance ratewas 99.0% (n = 387) at two digit resolution and 95.6% at four digitresolution.

2.4. Grouping of HLA alleles defined by KIR binding motifs

TheHLA class I alleles were groupedwith regard to their KIR bindingmotifs (for details, see: http://www.ebi.ac.uk/ipd/kir/ligand.html). ForHLA-A, the HLA-A*03 and HLA-A*11 encode ligands for KIR3DL2, andcarriers of these alleles were compared with non-carriers. TheHLA-C al-leles were divided into two epitope groups, C1 and C2. The HLA-C1group (*01, 03, 07, 08, 12, 13, 14, 16) encodes ligands for KIR2DL2 andKIR2DL3 and the HLA-C2 group (*02, 04, 05, 06, 15, 17, 18) encodes li-gands for KIR2DL1 and KIR2DS1. HLA-B was split into HLA-Bw4 andHLA-Bw6 groups based on serological epitopes defined by positions74–83 in their α1 domains. HLA-Bw6 is not known to bind KIRs, whileBw4 serves as a ligand for KIR3DL1 (Kaur et al., 2013).

2.5. Statistical analyses

HLA-allelic association testing and conditional logistic regressionanalyses were done using Unphased 3.0.10 (Dudbridge, 2008). Thedistribution of HLA class I alleles in relation to OCB status was condi-tioned on HLA-DRB1*15:01 and the distribution of KIR specificities inpatients and controls was conditioned on the HLA-DRB1 locus by thehaplotype methods (Valdes and Thomson, 1997). KIR binding motifswere analysed with chi square test using IBM SPSS version 20. A p-value b 0.05 was considered significant.

types in MS differ in their HLA class II association, while specific KIRoi.org/10.1016/j.jneuroim.2014.06.024

Table2

Distributionof

HLA

alleleslackingKIR

ligan

dmotifs

inOCB

nega

tive

MSpa

tien

ts,O

CBpo

sitive

MSpa

tien

tsan

dhe

althycontrols.

Lack

ofspecificKIR

ligan

dAllMSpa

tien

tsco

mbine

d,Observe

d/n(%

)OCB

nega

tive

MS

patien

ts,O

bserve

d/n(%

)OCB

positive

MS

patien

ts,O

bserve

d/n(%

)Co

ntrols,O

bserve

d/n(%

)OR(95%

CI)a

p-va

luea

OR(95%

CI)b

p-va

lueb

OR(95%

CI)c

p-va

luec

OR(95%

CI)d

p-va

lued

HLA

-A*03−

/−an

dHLA

-A*11−

/−13

89/255

9(54.3)

144/27

5(52.4)

1245

/228

4(54.5)

1954

/311

2(62.8)

0.70

(0.63–

0.78

)9.0E

−11

0.65

(0.51–

0.83

)0.00

10.71

(0.64–

0.79

)9.6E

−10

0.92

(0.71–

1.18

)0.50

HLA

–C1

−/−

235/25

45(9.2)

32/273

(11.7)

203/22

72(8.9)

378/31

12(12.1)

0.74

(0.62–

0.87

)4.6E

−4

0.96

(0.65–

1.41

)0.84

0.71

(0.59–

0.85

)1.8E

−4

1.35

(0.91–

2.01

)0.13

HLA

–C2

−/−

1239

/254

5(48.7)

128/27

3(46.9)

1111

/227

2(48.9)

1327

/311

2(42.6)

1.28

(1.15–

1.42

)6.0E

−6

1.19

(0.93–

1.52

)0.17

1.29

(1.16–

1.44

)5.0E

−6

0.92

(0.72–

1.19

)0.53

HLA

–Bw

4−/−

1368

/247

4(55.3)

150/26

3(57.0)

1218

/221

1(55.1)

1275

/297

4(42.9)

1.65

(1.48–

1.84

)6.5E

−20

1.77

(1.37–

2.28

)9.0E

−6

1.63

(1.46–

1.83

)3.1E

−18

1.08

(0.84–

1.40

)0.55

a:AllMSpa

tien

tsco

mbine

dve

rsus

controls.

b:OCB

nega

tive

MSpa

tien

tsve

rsus

controls.

c:OCB

positive

MSpa

tien

tsve

rsus

controls.

d:OCB

nega

tive

MSpa

tien

tsve

rsus

OCB

positive

MSpa

tien

ts.

3M.W. Gustavsen et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx

3. Results

3.1. Different KIR binding motifs in MS patients and healthy controls,irrespective of OCB phenotypes

The distributions of the KIR binding motifs in MS patients in total,OCB negative MS patients, OCB positive MS patients and healthy con-trols are shown in Table 2. A smaller proportion of MS patients lackedthe HLA-A*03 and HLA-A*11 alleles when compared to healthy controls(54.3% vs. 62.8%, OR= 0.70, 95% CI: 0.63–0.78, p= 9.0 × 10−11). Thesealleles encode molecules that are ligands recognized by the inhibitoryKIR3DL2. After conditioning on the HLA-DRB1 locus, the association be-came non-significant (p = 0.078). Moreover, the frequency of individ-uals lacking the C1 alleles, encoding ligands recognized by theinhibitory KIR2DL2 and KIR2DL3, was lower among the MS patientscompared with controls (9.2% vs. 12.1%, OR = 0.74, 95% CI: 0.62–0.87,p = 4.6 × 10−4). On the contrary, a higher proportion of the patientscompared to controls lacked the C2 alleles, which encode ligands forthe inhibitory KIR2DL1 and the activating KIR2DS1 (48.7% vs. 42.6%,OR = 1.28, 95% CI: 1.15–1.42, p = 6.0 × 10−6). Conditioning on theHLA-DRB1 locus yielded a marginally significant association for the C1and C2 allele groups (p = 0.045).The patients also more oftenlacked the alleles in the HLA-Bw4 group, which encode ligands forthe inhibitory KIR3DL1 (55.3% vs. 42.9%, OR = 1.65, 95% CI: 1.48–1.84,p=6.5 × 10−20). This association remained significant also after condi-tioning on HLA-DRB1 (p = 2.20 × 10−6). No significant difference be-tween the OCB negative and the OCB positive MS patients with regardto KIR binding motifs was observed (Table 2).

3.2. Shared effects of the HLA class I alleles in OCB negative and positiveMS patients

In the global analysis for HLA class I loci associations to different OCBsubtypes, only the HLA-A locus was significantly associated with OCBnegative MS compared to controls, in contrast to OCB positive MS,where all HLA class I loci showed differential overall allelic distributioncompared to controls (Table S1). When adjusting for the HLA-DRB1locus by conditional logistic regression analyses, the HLA-A locusshowed no significant association in the OCB negative MS patients(Table S1). For OCB positive MS patients, all HLA class I loci remainedsignificantly associated after conditioning on HLA-DRB1 (Table S1).The class I loci did not appear to differ significantly in their global alleledistribution between OCB positive and OCB negative MS (Table S1).The HLA allelic association testing showed that HLA-A*02:01protected against both OCB negative and positive MS (OR = 0.56, 95%CI: 0.46–0.69, p = 4.06 × 10−8 and OR = 0.67, 95% CI: 0.61–0.73,p = 3.87 × 10−21, respectively) (Table 3), and this remained signif-icant after conditioning on HLA-DRB1*15:01. In the unconditionedanalysis, HLA-B*44:02 was significantly less frequent in both OCBnegative and OCB positive MS patients compared to healthy con-trols, but this did not withstand conditioning for HLA-DRB1*15:01 in the OCB negative group (Table 3). HLA-C*05:01was significantly associated with OCB positive MS both in the un-conditioned and conditioned analyses (Table 3). Altogether, no sig-nificant difference was detected between the OCB negative andpositive MS patients with regard to HLA class I alleles (Table 3).

3.3. The HLA-DRB1 alleles are differently distributed in OCB negative andpositive MS patients

To replicate previous observations in a larger sample size, we in-vestigated the HLA-DRB1 alleles in relation to OCB phenotypes. Theglobal analyses showed that HLA-DRB1 was associated with both OCBpositive and OCB negative MS, but their significantly different allelicdistributions indicate that different HLA-DRB1 alleles are associatedwith different OCB phenotypes (Table S1). HLA-DRB1*15:01 was

Please cite this article as: Gustavsen, M.W., et al., Oligoclonal band phenotypes in MS differ in their HLA class II association, while specific KIRligands at HLA class I show associa..., J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.06.024

Table3

HLA

classIa

llelessh

owingassociationto

OCB

nega

tive

and/or

OCB

positive

MSpa

tien

tsco

mpa

redto

healthyco

ntrols.

Alle

lefreq

uenc

ies(%

)OCB

nega

tive

MSvs.con

trols

OCB

positive

MSvs.con

trols

OCB

nega

tive

MSvs.O

CBpo

sitive

MS

HLA

-alle

leOCB

-n=

275

OCB

+n=

2299

Controls

n=

3148

OR(95%

CI)

POR(95%

CI)*

P*OR(95%

CI)

POR(95%

CI)*

P*OR(95%

CI)

pOR(95%

CI)*

P*

HLA

-A*02:01

22.7

25.9

34.4

0.56

(0.46–

0.69

)4.06

E−

080.41

(0.25–

0.67

)0.00

043

0.67

(0.61–

0.73

)3.87

E−

210.64

(0.54–

0.76

)6.68

E−

070.84

(0.69–

1.04

)0.11

0.68

(0.42–

1.10

)0.12

HLA

–B*44

:02

7.0

7.2

11.0

0.62

(0.44–

0.87

)0.00

60.43

(0.16–

1.14

)0.08

90.67

(0.58–

0.77

)1.71

E−

080.63

(0.46–

0.88

)0.00

650.99

(0.70–

1.40

)0.94

0.57

(0.21–

1.55

)0.27

HLA

–C*05

:01

7.3

7.1

9.6

0.75

(0.54–

1.04

)0.09

0.58

(0.22–

1.53

)0.27

0.72

(0.62–

0.83

)3.51

E−

060.67

(0.47–

0.96

)0.03

01.05

(0.75–

1.47

)0.78

0.87

(0.33–

2.26

)0.77

*Con

dition

edon

HLA

-DRB

1*15

:01.

4 M.W. Gustavsen et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx

Please cite this article as: Gustavsen, M.W., et al., Oligoclonal band phenoligands at HLA class I show associa..., J. Neuroimmunol. (2014), http://dx.d

associated with both OCB positive and OCB negative MS whencompared with healthy controls (OR = 2.79, 95% CI: 2.56–3.04, p =2.82 × 10−118 and OR= 1.46, 95% CI: 1.22−1.75, p = 3.05 × 10−5, re-spectively) (Table 4). HLA-DRB1*04:04 conferred risk of OCB negativeMS (OR = 2.04, 95% CI: 1.58–2.63, p = 2.27 × 10−8), but not of OCBpositive MS. HLA-DRB1*04:01 was found to reduce the risk of OCB pos-itiveMS (OR= 0.66, 95% CI: 0.59–0.74, p= 6.61 × 10−13), but not OCBnegative MS. HLA-DRB1*01:01, *07:01, *13:01 and *13:02 were signifi-cantly associated with reduced risk for both OCB negative and OCB pos-itive MS (Table 4).

4. Discussion

This is the largest study to compare the frequencyof KIR bindingmo-tifs inMS patients comparedwith healthy controls and the first study toinvestigate HLA class I alleles and their KIR ligands in relation to OCBphenotypes in MS. With this extensive sample size, we observed thatOCB negative and positive MS patients did not carry different HLAclass I profiles or KIR binding motifs. However, our data confirm previ-ously described protective effects of the HLA-A*02:01, HLA-B*44:02and HLA-C*05:01 alleles in MS (Brynedal et al., 2007, Rioux et al.,2009, Link et al., 2010), supporting independent roles of HLA class I al-leles in the disease susceptibility.

When comparing the MS patients to healthy controls, the groupswere highly different with regard to KIR binding motifs. HLA-A3,which is recognized by the inhibitory KIR3DL2, has been shown to pre-dispose toMS (Harbo et al., 2004). In accordancewith this, we observeda lower frequency of patients than controls lacking the HLA-A*03 and*11 alleles encoding ligands for KIR3DL2 (Table 2). Moreover, it hasbeen described by us and others that KIR ligand HLA-Bw4 confers pro-tection against MS (Lorentzen et al., 2009, Garcia-Leon et al., 2011, Jelcicet al., 2012), and the present study further supports this observation.We found that HLA-Bw4, which is the KIR ligand for the inhibitoryKIR3DL1, was significantly reduced in MS patients compared to healthycontrols. This was highly significant also after conditioning on the HLA-DRB1 locus, indicating an effect independently of HLA-DRB1. This con-firms our findings in a Norwegian cohort with 631 MS patients and555 healthy controls (Lorentzen et al., 2009), in which there wereonly a small overlap with the samples included in the present study(17.0% of the cases and 7.3% of the controls). Furthermore, fewer pa-tients than controls lacked ligands for the inhibitory KIR2DL2 andKIR2DL3, which are encoded by alleles in the C1 allele group. TheKIR2DL2 has recently been implicated to influence the susceptibility toherpes virus infections inMS patients (Ben Fredj et al., 2014).Moreover,it has been suggested in a German study in 321 MS patients and 156healthy controls that absence of the inhibitory KIR2DL3 could be associ-ated with increased risk for MS in individuals carrying C1 alleles (Jelcicet al., 2012). In line with our observations, Jelcic and co-workers foundthat the frequency of individuals lacking C1 alleles tended to be lowerin the patients compared with controls. The activating KIR2DS1, whichrecognizes ligands from the C2 allele group, has been suggested tohave a protective role against MS, both in an Italian study comprising121 MS patients and 103 healthy controls (Fusco et al., 2010) and in aPortuguese study in 447 MS patients and 177 healthy controls(Bettencourt et al., 2014). This was supported by our findings of fewerMS patients having this KIR ligand. Thus, our observations replicate pre-vious suggested associations between KIR ligands and MS, and furthersupport the importance of NK cells and their ligands in the complex im-mune processes involved in MS.

NK cellsmight influence central and/or peripheral tolerance by elim-inating or modulating T regulatory cells or acting directly as regulatorycells (Poggi and Zocchi, 2014). It has been shown that HLA class I mole-cules are up-regulated on various cell types in the CNS, such as astro-cytes, oligodendrocytes, neurons and axons in active MS lesions(Hoftberger et al., 2004). Since NK cells interact with HLA class I mole-cules, they could potentially play an important role in the CNS of MS

types in MS differ in their HLA class II association, while specific KIRoi.org/10.1016/j.jneuroim.2014.06.024

Table 4HLA-DRB1 alleles showing association to OCB negative and/or OCB positive MS patients compared to healthy controls.

Allele frequencies (%) OCB negative MS vs. controls OCB positive MS vs. controls OCB negative MS vs. OCBpositive MS

HLA-allele OCB - n = 426 OCB + n = 3450 Controls n = 3148 OR (95%CI) p OR (95%CI) p OR (95%CI) p

HLA-DRB1*15:01 22.3 35.4 16.4 1.46 (1.22–1.75) 3.05E−05 2.79 (2.56–3.04) 2.82E−118 1.91 (1.61–2.26) 1.36E−13HLA-DRB1*13:02 3.4 4.0 5.5 0.62 (0.42–0.91) 0.015 0.72 (0.61–0.86) 0.0002 1.17 (0.79–1.72) 0.44HLA-DRB1*13:01 4.5 5.9 8.1 0.54 (0.38–0.76) 0.00037 0.71 (0.62–0.82) 2.43E−06 1.32 (0.94–1.86) 0.11HLA-DRB1*07:01 5.7 5.9 9.6 0.57 (0.42–0.78) 0.00034 0.59 (0.52–0.68) 2.51E−14 1.03 (0.76–1.41) 0.85HLA-DRB1*04:04 10.1 5.3 5.2 2.04 (1.58–2.63) 2.27E−08 1.02 (0.87–1.20) 0.81 0.50 (0.39–0.64) 5.71E−08HLA-DRB1*04:01 13.2 9.2 13.3 0.99 (0.80–1.23) 0.96 0.66 (0.59–0.74) 6.61E−13 0.66 (0.53–0.82) 0.0002HLA-DRB1*01:01 6.7 5.8 9.4 0.69 (0.52–0.93) 0.013 0.59 (0.52–0.68) 6.19E−14 0.85 (0.64–1.14) 0.30

5M.W. Gustavsen et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx

patients. Furthermore, NK cells have been implicated to have protectiveeffects in the animal model for MS, experimental autoimmune enceph-alomyelitis, by decreasing the local inflammation and autoimmune re-sponses against myelin antigens (Hao et al., 2010). Interestingly, it hasbeen suggested that the influence of regulatory CD56brigth NK cells onactivated T cells is the main mechanism of action for the humanizedmonoclonal antibody daclizumab, which is known to reduce CNS in-flammation in MS patients (Bielekova et al., 2006). Our data add to ac-cumulating evidence that NK cells are relevant in the pathogenesis ofMS. However, the biological effects of the KIR ligand associationswe ob-served are difficult to assess fully, since information about KIR geno-types was not available for the current cohort.

In addition to study HLA class I alleles and KIR binding motifs, weinvestigated HLA class II alleles in relation to OCB status. With 3876MS patients and 3148 healthy controls, we convincingly confirmedobservations from previous studies of HLA-DRB1 alleles in OCBnegative and positive MS patients reporting that the groups aregenetically distinct with regard to specific alleles (Kikuchi et al.,2003, Imrell et al., 2006, Idiman et al., 2009, Wu et al., 2010,Romero-Pinel et al., 2011, Mero et al., 2013). HLA-DRB1*04:04 alleleconferred risk of OCB negative MS, but not OCB positive MS. More-over, the major risk allele in MS, HLA-DRB1*15:01 conferred risk ofboth phenotypes, but appeared to confer a stronger risk of OCB pos-itive than OCB negative MS. This is in accordance with observationsin a Japanese study (nOCB+ = 53, nOCB- = 46) (Kikuchi et al., 2003), aSwedish study (nOCB+ = 184, nOCB- = 65) (Imrell et al., 2006) and arecent, large Scandinavian study (nOCB+ = 2781, nOCB- = 292)(Mero et al., 2013). In addition, we observed that HLA-DRB1*13:01, *13:02, *07:01 and *01:01 protected against both OCB nega-tive and OCB positive MS, while HLA-DRB1*04:01 only protectedagainst OCB positive MS, confirming our previous findings (Meroet al., 2013). We note that 9.0% of the cases and 18.3% of the controlsincluded in the present study were also included in the study ofMero et al.

It has been hypothesized that themore benign clinical disease man-ifestation and better therapy response observed in the OCB negativeMSpatients could be explained by a reduced T cell dependent activation ofB cells through presentation of peptides by HLA class II molecules in theOCB negativeMS patients (Lundkvist et al., 2010). This could potentiallybe influenced by variations in peptide binding abilities of different HLA-DRB1 molecules. Hence, our observation of a different distribution ofspecific HLA-DRB1 alleles, but not HLA class I alleles, in the OCB pheno-types, is biologically relevant. Further studies are needed to define theimmunological mechanisms involved.

A particular strength of this study is the large number of sampleswith information onOCB status collected from a representativeMS pop-ulation (Table 1), as lumbar puncture is routinely performed as a part ofthe diagnostic process in the Scandinavian countries. There are also po-tential limitations that should be kept inmindwhen interpreting the re-sults. First, the collection of samples has been done through severalyears and the methods for OCB detection have improved, with IEFbeing the most sensitive method and the AGE less sensitive (Awadet al., 2010). Some of the oldest samples analyzed with AGE could

Please cite this article as: Gustavsen, M.W., et al., Oligoclonal band phenoligands at HLA class I show associa..., J. Neuroimmunol. (2014), http://dx.d

have been falsely regarded as OCB negative. If this affects the results, itwould presumably lead to a weakening of the findings, rather thanfalse positive findings. Since the frequency of OCB negative patients inthe general MS population is low, the number of OCB negative MS pa-tients is a natural limiting factor when investigating OCB phenotypes.Even though the number of OCB negative MS patients included in thisstudy is relatively large (n = 426), true effects could be missed due tolack of statistical power. The fraction of OCB negative patients werelow in all the samples sets, but with some variation (Norway = 11.5%,Sweden = 8.9%, Denmark = 12.7%), thus these differences could notbe excluded as a bias. It should further be emphasized that majority ofthe HLA data included was imputed, and not obtained by a sequencebased approach, and a small fraction of the genotypesmight bewronglyimputed. Further, even though it previous has been shown that there islow population heterogeneity between the included cohorts (Meroet al., 2013), a potential confounding due to population differencescould influence the results.

5. Conclusions

This study is the first to investigate HLA class I alleles and their KIRbinding motifs in relation to OCB phenotypes in MS. Our findings indi-cate that the OCB positive and negative MS patients do not differ withregard to HLA class I alleles or KIR ligands.We also present an extensiveanalysis of the distribution of KIR binding motifs in MS patients com-pared to healthy controls, revealing significant differences betweenthe groups, supporting that activation or inhibition of NK cells throughKIRs could be involved in the pathogenesis of MS. Finally, we convinc-ingly confirm that the OCB negative and OCB positive MS patients arecharacterized by different HLA-DRB1 alleles, indicating that these phe-notypes are genetically distinct. Our observations underline the needfor further studies concerning the mechanisms behind the productionof OCBs in the CSF of the majority of MS patients.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jneuroim.2014.06.024.

Funding

This study was supported by a grant from the Research Council ofNorway (no: 196776), the Oslo MS Society, the Odd FellowMS Founda-tion, Knut and Alice Wallenberg Foundation, the Stockholm CountyCouncil, the Swedish Research Council, the Danish MS Society and theDanish Council for Strategic Research.

Acknowledgements

We thank all patients and controls for their participation. We thankThe International Multiple Sclerosis Genetics Consortium for imputingHLA information in the Norwegian and Danish samples. We also thankthe Oslo MS Society and Odd Fellow MS Foundation for support tofunding of the genotyping of the Norwegian samples. The NorwegianBone Marrow Donor Registry at Oslo University Hospital is acknowl-edged for providing Norwegian controls. We thank all contributors to

types in MS differ in their HLA class II association, while specific KIRoi.org/10.1016/j.jneuroim.2014.06.024

6 M.W. Gustavsen et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx

the collection of samples and clinical data in the NorwegianMS Registryand Biobank.

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