Cell-specific protein phenotypes for the autoimmune locus IL2RA using a genotype-selectable human bioresource

Abstract

Genome-wide association studies (GWAS) have identified over 300 regions associated with more than 70 common diseases. However, identifying causal genes within an associated region remains a major challenge. One approach to resolving causal genes is through the dissection of gene-phenotype correlations. Here we use polychromatic flow cytometry to show that differences in surface expression of the human interleukin-2 (IL-2) receptor alpha (IL2RA, or CD25) protein are restricted to particular immune cell types and correlate with several haplotypes in the IL2RA region that have previously been associated with two autoimmune diseases, type 1 diabetes (T1D) and multiple sclerosis. We confirm our strongest gene-phenotype correlation at the RNA level by allele-specific expression (ASE). We also define key parameters for the design and implementation of post-GWAS gene-phenotype investigations and demonstrate the usefulness of a large bioresource of genotype-selectable normal donors from whom fresh, primary cells can be analyzed.

Figure 1

The four common IL2RA/CD25 haplotypes and summary of the subject statistics by haplotype group. (a) The three independent T1D-associated SNP groups are located in the non-coding, 5′ and intron 1 regions of the IL-2RA gene on chromosome 10. The number of SNPs per associated group is 8, 3 and 2, as previously described, with each SNP being represented by a vertical bar-. Gray bars represent susceptibility alleles at these SNPs; the red, blue and orange bars represent protective alleles for each respective SNP group. The most associated SNPs in each group are rs12722495 (A>G), rs11594656 (T>A), and rs2104286 (A>G). The major alleles at all three SNPs are T1D susceptible and minor alleles are protective. Using the genotypes at these SNPs, four common haplotypes can be defined: “Susceptible”, “rs12722495”, “rs11594656”, and “rs2104286”. The frequency of these haplotypes in ≈9,000 healthy control individuals is shown, and the T1D association and odds ratios (OR) of the protective haplotypes were calculated relative to the ‘Susceptible’ haplotype using ≈7,000 case and ≈9,000 control samples (Supplementary Table 1). Chr, chromosome; kb, kilobases; Freq, frequency. (b) Statistics for the 179 individuals analysed in total. Individuals in the rs2104286 haplotype group did not have protective rs12722495 alleles. No individuals were heterozygous for two different protective haplotypes. SNP alleles are shown in the brackets. Hom, homozygotes; Het, heterozygotes; yr, year.

Figure 2

Gating strategy for CD4⁺ T-cell subsets. CD4⁺ T cells were gated based on their expression of IL-7R (CD127) and CD25 (inset plot shows isotype control staining). CD127^hi cells were further subdivided by their CD45RA expression, with CD45RA⁻ cells constituting the CD4⁺ memory T cell subset (14-76% of CD4⁺ T cells), and CD45RA⁺ cells constituting the CD4⁺ naive T cell subset (4-63% of CD4⁺ T cells). The CD127^int-low CD25^hi cells were gated on FOXP3 expression so as to define the FOXP3⁺ T-cell subpopulation (2-9% of CD4⁺ T cells). Representative CD25 expression profiles from individuals homozygous for susceptible alleles at all three SNPs (blue) and homozygous for rs12722495 protective alleles (and thus for rs2104286 protective alleles also because of the haplotype structure) (red) are shown for the CD4⁺ memory, naïve and FOXP3⁺ T cells. Samples from these individuals were obtained in the same donation session. Gray histograms are isotype control staining profiles. “Percent of max” is the cell number in each bin divided by the cell number in the bin containing the largest cell number; this statistic is calculated to normalize for different numbers of events collected for each sample that is overlaid. MFI, mean fluorescence intensity; MEF, molecules of equivalent fluorochrome.

Figure 3

IL2RA genotype and CD4⁺ T-cell phenotype correlations. (a) Individuals with at least one protective rs12722495 allele have higher CD25 expression on their CD4⁺ memory T cells (mean CD25 MEF=1,000) than fully susceptible individuals (mean=773) or protected rs11594656 (mean=810) or rs2104286 (mean=783) donors (1-d.f. P=4.3×10⁻⁹). (b) rs12722495 gene dosage effect on the CD25 MEF of CD4⁺ memory T cells (1-d.f. P=1.16×10⁻¹⁰). At rs12722495 A>G, where A=susceptible allele, G=protective allele. For AA, AG and GG donors mean CD25 MEF=788, 962 and 1,048, respectively. (c) Individuals with one or two protective rs2104286 alleles in the rs2104286 or rs12722495 haplotype groups, have a lower percentage of naïve cells that are CD25⁺ (for protective rs2104286 and rs12722495 donors mean=14 and 17%, respectively), compared to fully susceptible individuals or protected rs11594656 donors (mean=22 and 21% respectively; 1-d.f. P=1.6×10⁻³). (d) rs2104286 gene dosage effect on the percentage of CD4⁺ naive T cells that are CD25⁺ (1-d.f. P=4.25×10⁻⁶). At rs2104286 A>G, A=susceptible allele, G=protective allele. For AA, AG and GG donors mean percentage of CD4⁺ naïve cells that are CD25⁺=22, 20 and 11%, respectively. For the “combined” rs2104286 genotype, individuals with one or two protective alleles at this SNP are included regardless of allelic status at rs12722495. Susc., susceptible.

Figure 4

IL-2 secretion by stimulated CD69⁺ CD4⁺ T cell subsets. (a) Plot showing that unstimulated CD4⁺ memory T cells do not upregulate CD69 at their cell surface and do not secrete IL-2. (b) Upon stimulation of isolated PBMCs for 4 hours with 5 μg/ml of staphylococcal enterotoxin B (SEB), a proportion of CD4⁺ memory T cells became CD69⁺ and secreted IL-2. (c) Eight pairs of previously immunophenotyped donors were recalled such that in each pair one donor was homozygous for the protective rs12722495 haplotype and one donor was homozygous for the fully susceptible haplotype (and had CD4⁺ memory T cells with CD25 levels <800 MEF). Susceptible donors were selected to have low CD25 levels on their CD4⁺ memory cells in order to test the hypothesis that differential CD25 expression results in differential IL-2 production. A higher proportion of the CD69⁺ CD4⁺ memory T cells from protective donors secrete IL-2 compared to fully susceptible donors (P=5.74×10⁻⁴). The average difference in CD25 levels of the unstimulated CD4⁺ memory T cells between the protective and susceptible donors (23.2%) corresponds to the average difference in IL-2 secretion observed for these donors (22.5%). (d) A genotypic correlation was not observed for the proportion of CD69⁺ CD4⁺ naïve T cells secreting IL-2 (P=0.318).