State-specific effects of eQTL SNPs.

<p><b>A</b>) For a subset of genes, the correlation effects (β) of the top associated SNP across resting and stimulated cells differed. The genes shown with a black-dotted vertical lines had significantly different effect sizes across states. Black horizontal segments in <b>B</b>)<b>–D</b>) denote median values. Blue panels show resting-state (normalized) expression values; red panels show stimulated expression values. <b>B</b>) rs942793^G significantly increased the expression of <i>ZMIZ1</i> only in stimulated cells. <b>C</b>) rs12746918^T was correlated with increased expression of <i>PLCH2</i> in resting cells only. <b>D</b>) rs4840565^C decreased <i>BLK</i> expression in stimulated cells nearly twice as much as in resting cells [<i>β</i>_rest(SE) = −0.366(0.085), <i>β</i>_stim = −0.805(0.071)].</p

Schematic of the experimental workflow.

<p>We collected four types of data from each individual: 1) quality-controlled genome-wide SNP data containing 638,347 markers collected on Illumina Infinium Human OmniExpress Exome BeadChips, 2) abundance of CD4 T_EM cells as a percentage of all CD4 T cells obtained by FACS and quantified by X-Cyt, 3) average cell division upon T cell receptor stimulation by anti-CD3/CD28 commercial beads, measured using a CFSE (carboxyfluorescein succinimidyl ester) dye dilution assay, and 4) expression of 215 genes measured by NanoString nCounter. We repeated each proliferation assay in two-three technical replicates. Cell sorting purity and replication correlations for CD4 T_EM abundance, division index, and proliferation index are shown in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004404#pgen.1004404.s002" target="_blank">Figure S2</a></b>.</p

Five disease risk alleles explained the eQTL associations with five genes.

<p>The left-sided panels show unconditional SNP-expression association results. Green dashed lines mark the TSS of the eQTL gene. The red dots indicate the risk alleles associated with the expression of respective genes shown as red arrows. The right-sided panels show adjusted association results after conditioning for the respective risk alleles. In each of the five loci, conditioning on the disease SNP obviated signals in the entire region, such that no association more significant than <i>p</i> = 0.05 remains.</p

Genome-wide association to division index (the average number of division undergone by all cells).

<p><b>A</b>) rs389862^A on chromosome 13 was significantly associated to increased division index at <i>p</i> = 4.75×10⁻⁸, and is located in a non-coding region 30 kb upstream of <i>RASA3</i>, and 70 kb upstream from <i>CDC16</i>. <b>B</b>) rs3775500^G on chromosome 4 shows a strongly suggestive association at <i>p</i> = 5.40×10⁻⁷, and is located within the <i>DAPP1</i> (Bam32) gene.</p

Relationship between baseline expression and post-stimulatory response.

<p><b>A</b>) Baseline expression of 17 genes correlated with post-stimulation proliferation. Rows in the heatmap are ordered from top to bottom by ascending proliferation index. Genes/columns are ordered from the most negatively correlated (IL23RB) to the most positively correlated (CCR9). The 17-gene signature was calculated as the weighted sum of the 17 genes, where the negatively-correlated genes were given a weight of −1, and the positively-correlated genes were given a weight of +1. <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004404#pgen.1004404.s008" target="_blank">Table S1</a></b> lists the correlation coefficient and p-value for each gene. <b>B</b>) Genes correlated with proliferative response were enriched for apoptosis and lymphocyte activation pathways. Genes correlated to lower proliferative response (proliferative index) were enriched for Gene Ontology code GO:0012502 (induction of programmed cell death, p = 1.8×10⁻⁴). Conversely, genes correlated to higher proliferative response were enriched for GO:0002285 (lymphocyte activation, p = 3.9×10⁻⁴).</p

<i>Cis</i>- associations detected with Spearman Rank Correlation analysis of normalized and PCA-corrected expression data.

<p><i>Cis</i>- associations detected with Spearman Rank Correlation analysis of normalized and PCA-corrected expression data.</p

Forest plot of component effects of complete GWAS predictive model based on training set of SNPs.

<p>Odds ratios (black squares) from the complete multivariate model (“chromstate+eqtl [M3]”) for features predicting the membership of a SNP in the NHGRI GWAS Catalog are shown here with standard errors (gray lines). Smaller models are shown for comparison in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140758#pone.0140758.s002" target="_blank">S2 Fig</a>. Four classes of SNP annotation are represented in the model, each with multiple levels: distance from gene, MAF, chromatin state in GM12878 LCLs (12), and evidence of eQTL association based on meta-analysis FDR. The base levels for each annotation are “0 kb (within gene)” [Distance from Gene], “>10%” [MAF], “Heterochromatin (13)” [ChromHMM], and “>50%” [FDR].</p

Cell-state specific eQTLs.

<p>For each row we list a SNP and the gene transcript for which it is a <i>cis</i>-eQTL. We indicate whether the effect is observed in resting or stimulated CD4+ effector memory T cells. For each SNP we indicate the fold change in expression conferred per allele, assuming an additive model and the false discovery rate estimate. Finally, we indicate whether the effect is specific for CD4+ effector memory T cells; we define specificity as the absence of any cis-eQTL effect in PBMCs at FDR>50% <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004404#pgen.1004404-Westra1" target="_blank">[8]</a>.</p><p>* pΔ measures the difference between the effect sizes across resting and stimulated states.</p

Risk alleles of CeD, RA, and T1D, showed no significant association to CD4 T_EM cell abundance or proliferation.

<p><b>A</b>) The 118 SNPs with association to diseases in densely genotyped regions on Immunochip platform were not significantly associated to CD4 T_EM cell abundance. The shaded region shows 95% confidence interval. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004404#pgen.1004404.s005" target="_blank">Figure S5</a></b>. <b>B</b>) The same set of 118 risk alleles also showed no inflation in association with proliferative response measured as division index.</p

Expression level fold-change for significant SNP-probe <i>cis</i>- associations shared by pairs of populations.

<p>Shown are plots of the absolute value of expression level fold-change between median expression levels of homozygote classes for significant associations (permutation threshold 0.01) for each pairwise combination of populations. Within a panel, deviating from the 1 to 1 line (lower left to upper right) indicates differences in expression level fold-change (effect size) on log₂ scale in the two populations being compared.</p