the important role of variation in genes of the human leukocyte antigen ( hla ) complex in genetic predisposition to multiple sclerosis ( ms ) has long been recognized . however , in addition to their role in antigen presentation to t cells , hla class i molecules also serve as ligands for kir molecules . an ever - increasing pace of investigation over the last decade has demonstrated definitively that killer immunoglobulin - like ( kir ) receptors play critical roles in transplantation success and disease pathogenesis ( 14 ) . expressed on the surface of natural killer ( nk ) cells , kir serve to mediate cytolytic killing and cytokine secretion ( 5 ) . the kir gene complex on human chromosome 19q13.4 encodes both inhibitory and activating receptors , with significant variation in gene content between individuals and populations . any given kir haplotype may contain from 414 genes , and are generally categorized into two groups , termed a and b ( 6 ) . kir molecules recognize specific epitopes on hla class i : kir3dl2 and kir2ds4 recognize hla - a molecules with the a3/a11 epitope , while kir2dl1 , kir2dl2/3 , kir2ds1 , kir2ds2 and kir2ds4 interact with either the c1 or c2 epitopes on hla - c ; a small subset of hla - b molecules also carry the c1 epitope , and are capable of interacting with these kir . finally , kir3dl1 binds hla - a and -b molecules that carry the bw4 specificity ( 2 , 79 ) . only a handful of studies have been conducted examining kir variation in ms , but overall , the analysis of kir gene - content variation has pointed to a role for the kir in disease susceptibility . in patients of european ancestry , predisposition to ms has been variably associated with the absence of the inhibitory kir2dl3 ( 10 ) or the presence of activating kir2dl5 and kir3ds1 . in other studies , kir2ds1 was found to be protective in ms ( 11 , 12 ) . in the largest previous study to - date , the hla class i bw4 motif was found to be protective in a norwegian cohort , but no association was observed between kir carrier frequencies and ms ( 13 ) . all prior examination of the role of kir in ms has been conducted in cohorts of european origin . while conventional wisdom has held that ms is much less common in african americans relative to european americans , more recent data suggests that risk for ms in african americans is higher than expected and its incidence in this group is increasing ( 14 , 15 ) . importantly , african americans are more likely to have a more acute disease course and it appears that this increased severity is partially associated with african ancestry ( 16 , 17 ) . examination of hla associations with ms in african americans has served to clarify hla class ii haplotypic associations ( 18 ) and revealed a role for hla - drb5 ( 19 ) . here , we investigate the role of the kir loci and their hla class i ligands in a large cohort of african american ms patients and controls . in addition to being the first investigation of kir and autoimmune disease in african americans , this study represents the largest case - control analysis conducted to - date in any population examining kir and hla variation in ms . no significant difference in carrier frequencies for any kir locus ( or allele in the case of kir2dl2/3 and kir3dl1/s1 ) , were observed between cases and controls ( table 1 ) . we also examined whether specific haplotypic structures of the kir were associated , rather than individual loci , both across the entire kir cluster and within each of the centromeric and telomeric regions . no significant associations were observed for any specific kir gene - content haplotype or particular combinations of kir a and b haplotypes ( data not shown ) . however , examination of kir in the context of their cognate hla ligands ( table 2 ) revealed a strong protective effect for kir3dl1 in combination with hla - a and b alleles bearing the bw4 motif ( p=10 ; or=0.60 , ci=0.500.71 ) . because nearly every individual in this cohort is positive for kir3dl1 ( table 1 ) , a protective effect is also expected and observed for the bw4 ligand alone ( table 3 ; p<10 ; or=0.63 , ci=0.530.75 ) . these results are in keeping with those observed in the norwegian cohort ( 13 ) , where , as in most populations ( 20 ) the carrier frequency of kir3dl1 is also very high . we tested all hla - b alleles for association with disease in order to determine whether the bw4-mediated effect could be attributed to one or a few specific hla - b alleles . only one hla - b allele , hla - b*53 would be considered significantly protective in this cohort after bonferroni correction for multiple tests ( < 0.002 ) . while this allele does have the bw4 motif , it is not present at sufficient frequency in this cohort ( < 4% in cases and controls ; supplemental table 1 ) to account for the observed bw4 association . we therefore considered whether the observed association with bw4 could be attributed to linkage disequilibrium of bw4-bearing hla - b alleles with specific ms - associated hla alleles at other loci . when two locus haplotypes ( hla - b~hla - drb1and hla - a~hla - b ) are considered with hla - b alleles coded for either the bw4 or bw6 motif , in each case the most significantly protective haplotype bears a bw4 hla - b allele , while the hla - a and hla - drb1 alleles vary ( table 4 ) . for example , when hla - drb1 is considered alone , protection is conferred by the common allele hla - drb1 * 11 ( p=0.0001 ; or=0.69 , ci=0.570.84 ) . likewise , the most highly significant protective hla - drb1~hla - bw4/6 haplotype is hla - drb1 * 11~bw4 ( p<106 ; or=0.50 , ci=0.340.72 ) . in contrast , the hla - drb1 * 11~bw6 haplotype is not significantly associated with disease . similarly , the two other haplotypes observed to be protective in this cohort are hla - drb1 * 13~bw4 ( p<0.05 ; or=0.67 , ci=0.490.93 ) and hla - drb1 * 07~bw4 ( p<0.05 ; or=0.58 , ci=0.400.85 ) , but their bw6-bearing counterparts are not . in each of these cases , the bw4 and bw6 bearing hla - drb1 haplotypes are present at roughly equal frequencies , thus ruling out linkage disequilibrium as a source of the disparate effects . when we consider common ( frequency > 0.03% ) haplotypes with hla - a , only hla - a*02~bw4 is significantly protective in this cohort ( p=0.003 ; or=0.70,ci=0.550.88 ) . when hla - a is considered alone ( supplemental table 1 ) , hla - a*02 is not protective , nor is it protective in the context of bw6 rather than bw4 bearing hla - b alleles ( table 4 ) . taken together , the haplotype data indicate that the protective effect is being mediated by the presence of the bw4 epitope that confers kir3dl1 ligand status to hla - b alleles . the observed effect can not be explained by either a specific hla - b allele defined with respect to the antigen - binding domain ( at single - field resolution ) or by any protective hla - drb1 or hla - a alleles in linkage disequilibrium with bw - bearing hla - b alleles , supporting the relevance of the kir - hla framework in disease risk . the strong predisposing effect of hla - drb1 * 15:01 in ms has long been recognized , and in this cohort hla - drb1 * 15:01 , like nearly most others examined to - date ( 21 ) , is the primary predisposing hla variant with or>2 ( supplemental table 1 ) . given the strong and significant role for hla - drb1 * 15:01 , we explored whether protection mediated via kir3dl1/hla - bw4 is differentially associated with regard to hla - drb1 * 15:01 status . cases and controls were stratified according to having 0 , 1 or 2 copies of hla - drb1 * 15:01 , and association of kir3dl1/bw4 with disease was examined within each stratum ( table 5 ) . the protective effect was observed only in individuals who were not positive for hla - drb1 * 15:01 ( p<10 ; or=0.61 , ci=0.510.74 ) . these data suggest that while the kir - ligand combination mediates a robust protective effect , that effect is overridden in the presence of the strong predisposing effect of hla - drb1 * 15:01 . analysis of hla - b~hla - drb1 haplotypes in hla - drb1 * 15:01-negative individuals yields results similar to those described above for the entire cohort ( data not shown ) , implying that differential association of kir3dl1/bw4 is not mediated by linkage disequilibrium between hla - drb1 and hla - b alleles . given the extensive allelic polymorphism of kir3dl1 , future studies examining kir in ms will benefit from high - resolution genotyping , particularly in populations with african ancestry . for example , a non - expressed variant , kir3dl1 * 004 ( 22 ) is observed at relatively high frequencies in a west african population ( 23 ) . it is also interesting to note that kir3dl1 and hla - bw4 were found to be subject to co - evolution in this same population , suggesting a selective advantage for this kir - hla combination . in conclusion , our data in a large african american cohort confirm and refine a previous finding in a large norwegian cohort ( 13 ) of protection from ms mediated by the kir3dl1 ligand hla - bw4 , either alone or in combination with kir3dl1 . the fact that our results in a large african american cohort also implicate bw4 , despite the fact that individuals with european and african ancestry have vastly different hla - b allelic variation and frequency distributions ( 24 ) , supports the notion that the functional properties of the bw4 motif with respect to kir3dl1 and nk cell inhibition and/or licensing are the key determinants in protection from disease . the study cohort consisted of 907 african american ms cases and 1456 african american controls . ascertainment protocols and clinical and demographic characteristics have been summarized elsewhere ( 16 , 27 ) . principal component analysis and pruned genome - wide autosomal non - mhc snps with minor allele frequency ( maf ) > 1% were used to assess ancestry and control for the effects of population stratification ( 28 ) . dna samples were collected from patients and kir typing was performed for presence or absence of kir genes by the labtype sso kir typing ( onelambda , inc . the typing system can distinguished 8 inhibitory genes ( kir2dl1 - 2dl5 , kir3dl1 - 3dl3 ) , 6 activating genes ( kir2ds1 - 2ds5 , kir3ds1 ) and 2 pseudogenes ( kir2dp1 and kir3dp1 ) . genotypes for hla - drb1 , hla - a , -b and c were obtained by ssop using the labtype sso hla typing ( onelambda , inc . all statistical analysis was performed using the r language for statistical computing ( 29 ) . all single - locus tests for association were performed using standard chi - squared analysis with the odds ratios and confidence intervals were calculated using the  epitools package for r ( 30 ) . in the case of low - frequency cells , as is common in hla data , alleles with expected counts < 5 were combined into a single  binned  category prior to analysis ( 31 ) . haplotype estimation and association tests were performed using the  haplo.stats package ( 32 , 33 ) . dna samples were collected from patients and kir typing was performed for presence or absence of kir genes by the labtype sso kir typing ( onelambda , inc . the typing system can distinguished 8 inhibitory genes ( kir2dl1 - 2dl5 , kir3dl1 - 3dl3 ) , 6 activating genes ( kir2ds1 - 2ds5 , kir3ds1 ) and 2 pseudogenes ( kir2dp1 and kir3dp1 ) . genotypes for hla - drb1 , hla - a , -b and c were obtained by ssop using the labtype sso hla typing ( onelambda , inc . all statistical analysis was performed using the r language for statistical computing ( 29 ) . all single - locus tests for association were performed using standard chi - squared analysis with the odds ratios and confidence intervals were calculated using the  epitools package for r ( 30 ) . in the case of low - frequency cells , as is common in hla data , alleles with expected counts < 5 were combined into a single  binned  category prior to analysis ( 31 ) .
we investigated the role of the kir loci and their hla class i ligands in a large cohort of african american ms patients ( n=907 ) and controls ( n=1456 ) . 
 no significant differences in carrier frequencies for any kir locus or haplotype were observed between cases and controls . 
 however , examination of kir in the context of their cognate hla ligands revealed a strong protective effect for kir3dl1 in combination with hla - a and b alleles bearing the bw4 motif ( p=108 ; or=0.60 , ci=0.500.71 ) and the bw4 ligand alone ( table 3 ; p<106 ; or=0.63 , ci=0.530.75 ) . the observed effect can not be explained by either a specific hla - b allele or by linkage disequilibrium with hla - drb1 or hla - a . 
 the protective effect was observed only in individuals who were not positive for the ms risk allele hla - drb1 * 15:01 ( p<106 ; or=0.61 , ci=0.510.74 ) . 
 our study , the first investigation of kir and ms in african americans , confirms and refines previous findings in a european cohort .