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. 2014 Mar 25;111(12):E1111-20.
doi: 10.1073/pnas.1401343111. Epub 2014 Mar 7.

Interindividual variation in human T regulatory cells

Affiliations

Interindividual variation in human T regulatory cells

Alessandra Ferraro et al. Proc Natl Acad Sci U S A. .

Abstract

FOXP3(+) regulatory T (Treg) cells enforce immune self-tolerance and homeostasis, and variation in some aspects of Treg function may contribute to human autoimmune diseases. Here, we analyzed population-level Treg variability by performing genome-wide expression profiling of CD4(+) Treg and conventional CD4(+) T (Tconv) cells from 168 donors, healthy or with established type-1 diabetes (T1D) or type-2 diabetes (T2D), in relation to genetic and immunologic screening. There was a range of variability in Treg signature transcripts, some almost invariant, others more variable, with more extensive variability for genes that control effector function (ENTPD1, FCRL1) than for lineage-specification factors like FOXP3 or IKZF2. Network analysis of Treg signature genes identified coregulated clusters that respond similarly to genetic and environmental variation in Treg and Tconv cells, denoting qualitative differences in otherwise shared regulatory circuits whereas other clusters are coregulated in Treg, but not Tconv, cells, suggesting Treg-specific regulation of genes like CTLA4 or DUSP4. Dense genotyping identified 110 local genetic variants (cis-expression quantitative trait loci), some of which are specifically active in Treg, but not Tconv, cells. The Treg signature became sharper with age and with increasing body-mass index, suggesting a tuning of Treg function with repertoire selection and/or chronic inflammation. Some Treg signature transcripts correlated with FOXP3 mRNA and/or protein, suggesting transcriptional or posttranslational regulatory relationships. Although no single transcript showed significant association to diabetes, overall expression of the Treg signature was subtly perturbed in T1D, but not T2D, patients.

Keywords: immunoregulation; suppression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Interindividual variability in Treg signature transcripts. (A) Gene-expression profiles were generated from purified Treg and Tconv cells from blood of 168 individuals, and the average expression values are compared. (B) Treg signature transcripts are ranked according to mean differential expression (red dots), and the Treg/Tconv ratio for each individual of Cohort3 is plotted. (C) Normalized microarray expression levels in Tconv and Treg cells of selected genes (on a linear scale from 20 to 20,000, where 120 represents a 95% probability of expression); each dot represents 1 of 55 donors from cohort 3. (D) Histogram of gene-wise coefficient of variation across the datasets for Treg signature genes or for a random expression-matched set of genes (P value, Wilcoxon rank-sum test).
Fig. 2.
Fig. 2.
Coregulated gene clusters in Treg and Tconv cells. (A) Experimental concept. (B) Treg signature genes were tested for correlated expression across all donors, and the 290 genes with >2 correlated genes (at cc > 0.5) were biclustered within the Treg and Tconv expression datasets. Heatmap representation of the gene–gene correlation coefficients, similarly clustered in the Treg and Tconv datasets. The Cluster Similarity score is the mean of Pearson correlation coefficients between members of each cluster, computed in Treg and Tconv cells. Members of the Treg-specific clusters are shown.
Fig. 3.
Fig. 3.
eQTL analysis in Treg and Tconv gene expression. (A) Genome-wide SNP genotypes from 57 individual belonging to cohorts 2 and 3 were tested for association to expression of neighboring genes in the Treg and Tconv datasets. The significance of the association is shown [−log10(P value)]; each dot is an SNP/gene pair. The dotted line denotes the nominal P value for significance. (B) Comparison of maximum eQTL P values for genes in Treg and Tconv expression datasets. Red or blue dots and labels are Treg-Up and -Down signature genes, respectively. (C) eQTL P values are plotted against the Variability Score (measure of true interindividual variability, after correcting for experimental noise).
Fig. 4.
Fig. 4.
Bias in Treg gene expression with age. (A) Gene expression values in the Treg dataset were fitted in a mixed-model with donor age, sex, and BMI as explanatory variables, independently for T1D and healthy controls, and the coefficients of the fit were plotted (P values were computed from a χ2 test of the distribution of positive and negative coefficients). (B) A “Treg index” was calculated for each individual (by averaging normalized expression values of all genes in the Treg-Up or -Down signatures) and plotted against the donor age, for Treg and Tconv cells.
Fig. 5.
Fig. 5.
Relationships to FOXP3 expression and Treg function. (A) Expression of individual genes was correlated to Treg frequency (proportion of FOXP3+CD25+ cells within CD4+ cells), and the correlation coefficients were plotted (blue and red dots, Treg-Up and -Down signature genes, respectively). (B) Plot of the coefficients against the Treg/Tconv expression ratio. (C and D) As in A and B, but vs. FOXP3 mean fluorescence intensity in CD4+CD25+CD127lo cells. (E) Correlation of all genes to FOXP3 mRNA expression in the Treg dataset vs. correlation to FOXP3 protein [from flow cytometry MFI; blue and red dots, Treg-Up and -Down signature genes, gated on FoldChange > 2)]. (F) Correlation of gene expression vs. Treg suppressor activity, measured in a subset of 22 donors. (G) Ranked plot of correlation coefficients for all genes (black dots) or for the Treg-Up and -Down signature genes (red, blue).
Fig. 6.
Fig. 6.
Underexpression of Treg signature genes in Treg cells from T1D patients. (A) Expression values of each gene in the Treg and Tconv datasets were compared in Tregs from T1D patients or healthy controls. The nominal threshold for corrected genome-wide significance is shown as a dotted line. (B) Mean Fold Change vs. t test P value for the comparison between T1D and healthy donors for Treg-Up (Left) and -Down (Right) signature genes. (C) As in B, comparing T2D and healthy donors.

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