Frequency of expression of KIR3DL1/S1, KIR2DS4 and KIR2DL3 on the NK cell population.

<p>(A) The percentage of NK cells expressing KIR3DL1 in donors containing only one expressed inhibitory allele is indicated for individuals with a <i>KIR3DS1/3DL1</i> genotype. (B) The percentage of NK cells expressing either KIR3DS1 or the indicated KIR3DL1 allotypes was determined in individuals heterozygous for <i>KIR3DS1</i> or <i>KIR3DL1</i> and the <i>KIR3DL1*004</i> allele (*004 is not expressed on the NK cell surface). (C) The percentage of donor peripheral blood NK cells expressing KIR2DS4 is shown. Individuals are grouped based on the presence of the expressed allele or the non-expressed <i>KIR2DS4</i> alleles that possess a 22 bp deletion (<i>KIR2DS4</i>-del) Heterozygous <i>KIR2DS4*001/KIR2DS4</i>-del donors possess one expressed <i>KIR2DS4</i> allele, whereas <i>KIR2DS4*001</i> (in the absence of the null allele) individuals are expected to possess one copy of <i>KIR2DS4</i> but could potentially contain a second <i>KIR2DS4*001</i> allele. (D) Expression of KIR2DL3 on peripheral blood NK cells is shown. Individuals typed as possessing <i>KIR2DL3</i> virtually always have two copies of the gene, whereas donors that have <i>KIR2DL2/2DL3</i> are expected to have only a single copy of <i>KIR2DL3</i>.</p

Organization of the <i>KIR</i> gene cluster.

<p>The <i>KIR</i> gene order is shown for the A haplotype and a representative B haplotype. Inhibitory KIR genes are shown as red boxes, whereas the activating genes are shown in green. The framework genes are indicated by grey boxes, and pseudogenes are indicated by black boxes. Each gene spans between 10–16 kb, and the intergenic distances are approximately 2 kb with the exception of the 14 kb region upstream of the <i>KIR2DL4</i> gene.</p

Quantitative analysis of <i>KIR3DL1/S1</i> antisense transcripts.

<p>(A) Comparison of relative antisense transcript levels in individuals bearing alleles shown to have high (*002) or low (*001/*004) antisense promoter activity <i>in vitro</i>. (B) Correlation of relative antisense transcript levels with frequency of NK cells expressing different KIR3DL1 allotypes. Symbols shown correspond to individuals possessing the <i>KIR</i> alleles as shown in A. The <i>KIR3DL1*004</i> allele was removed from the analysis since it is not expressed on the NK cell surface.</p

Impact of polymorphisms present in the <i>KIR3DL1</i>, <i>KIR2DS4</i>, <i>KIR2DL3</i>, and <i>KIR2DL5</i> genes on promoter activity.

<p>(A) A schematic of the <i>KIR</i> bi-directional promoter region is shown with the position of transcription factor binding sites indicated by labeled boxes. The sites of transcript initiation for the sense and antisense promoters are shown by the rightward and leftward arrows respectively. The vertical lines indicate the positions of polymorphic nucleotides relative to the <i>KIR3DL1*002</i> allele. Numbers above the lines indicate the positions of the polymorphic residues relative to the start codon of the <i>KIR3DL1</i> gene. The nucleotide present at each variable position is shown for the <i>KIR3DL1*002</i> gene and only differences are shown for the remaining alleles. (B) The promoter activities of <i>KIR3DL1/S1</i> promoter alleles are shown. The 229 bp core promoter region of the <i>KIR3DL1</i> alleles and <i>KIR3DS1</i> were cloned into the pGL3 reporter vector in the forward and reverse orientation, and the promoter activity of each construct was determined by transfection into the YT-Indy human NK cell line. The ratio of forward promoter activity to reverse activity (F/R ratio) is listed for each allele. (C) Promoter activities of <i>KIR2DL5</i> alleles. (D) Characterization of promoter activity of the <i>KIR2DS4</i> alleles and the <i>KIR2DL3</i> promoter. Values represent fold increase of luciferase activity relative to empty pGL3 vector. The mean and SD of at least 3 independent experiments are shown.</p

Effect of <i>KIR</i> promoter polymorphisms on Sp1 binding.

<p>EMSA analysis of the Sp1 binding region corresponding to the polymorphisms observed in <i>KIR</i> alleles. (A) Oligonucleotides used for EMSA. The sense strand of oligonucleotide probes corresponding to the predicted Sp1-binding region of the indicated <i>KIR</i> genes is shown. The nucleotide residue labeled (−26) corresponds to the same −26 position shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000254#pgen-1000254-g002" target="_blank">Figure 2A</a>. (B) EMSA analysis performed on YT nuclear extracts with probes indicated in A. The right panel shows a supershift of the Sp1 consensus probe from YT extracts or in the presence of recombinant human Sp1 protein (rhSp1).</p

Four-loci, dbMHC Irish data.

<p>Abbreviations are: S = softmax, RM = regularized multinomial, M = non-regularized multinomial, AM = allele marginals. A total of 468 alleles were masked in the test set.</p

Ability of p24 Gag_209–218 peptide variants for HLA-Cw*0102 stabilization and KIR2DL2-Fc binding.

<p>(<b>A</b>) Differential expression of HLA-Cw*0102 on T2 cells after co-incubation with peptide variants of HIV-1 p24 Gag_209–218-L (AAEWDRLHPV). Peptide variants differed in length [9 or 10 amino acids (aa)] as well as in sequence (substitution of Leucine in position 7 with various amino acids). HLA-Cw*0102 expression is illustrated as relative median fluorescence intensity (RFI) as compared to unloaded T2 cells. Each column represents mean±SEM RFI of 4 independent experiments for each peptide variant. (<b>B</b>) Relative binding of KIR2DL2-Fc to T2 cells after co-incubation with selected HIV-1 p24 Gag_209–218variants. KIR2DL2-Fc binding is illustrated as RFI as compared to VAP-DA-pulsed T2 cells. Each column represents mean±SEM RFI of 3 independent experiments for each 10 aa variant. (<b>C</b>) Different levels of NK cell degranulation after co-incubation with T2 cells in the presence of different p24 Gag_209–218-L variants. Each column represents mean±SEM percentage of CD107a(+) NK cells of 4 different individuals. Unspecific degranulation of NK cells measured without target cells was deducted from each column. * indicates statistical significance as defined <i>p</i><0.05.</p

Summary of Private Data

<p>Summary of Private Data</p

p24 Gag_209–218/HLA-Cw*0102 complexes on T2 cells lead to functional inhibition of primary KIR2DL2(+) NK cells.

<p>(<b>A</b>) Representative histograms of degranulation of KIR2DL2(−) (left panel) and KIR2DL2(+) (right panel) NK cells after co-incubation with peptide-pulsed T2 cells (tinted and black) or without target cells (clear). NK cell degranulation was measured flow cytometrically by surface expression of CD107a. (<b>B</b>) Different levels of NK cell degranulation between KIR2DL2(−) (dark grey) and KIR2DL2(+) (light grey) NK cells after co-incubation with peptide-pulsed T2 cells. Each column represents mean±SEM percentage of CD107a(+) NK cells of 5 of different individuals. Unspecific degranulation of NK cells measured without target cells was deducted from each column.</p

Statistical Significance Results on Private Data, Separately for Each Ethnicity.

*<p>Denotes the method that performed better (except for the last row, where the allele marginals perform better than the unregularized multinomial on the log likelihood, but worse on the number of correct MAP predictions.</p