Figure 1.
A. CD4+CD25- T cells were sorted by flow cytometry into CD45RO- and CD45RO+ subsets. Memory CD4+CD25-CD45RO+ cells were sorted into CD161+ CCR6+ and CD161-CCR6- subsets for subsequent activation and expansion. B. Th1 cells were generated from naïve CD4+CD62L+CD25-CD45RO- T cells by activating and expanding with anti-CD3, anti-CD28 in the presence of IL-12 and anti-IL4 for seven days. CD161+CCR6+ and CD161-CCR6- memory CD4+ T cells were activated and expanded with anti-CD3, anti-CD28, IL-1β and IL-23 for seven days. The distinct T cell subsets were assessed for differentiation by intracellular cytokine staining for IL-17A and IFNγ expression. Representative flow cytometry plots are shown in the figure.
Figure 2.
Improved power to identify differential gene expression with RNAseq.
A. Q-Q plot for observed and expected P-Values for differential gene expression comparing in vitro differentiated Th1 and Naïve CD4+ T cells (N = 4). B. Observed statistical significance for differential gene expression between in vitro differentiated Th1 and Naïve CD4+ T cells (N = 4). C. Q-Q plot for observed and expected P-values for differential gene expression comparing Th17-enriched and Th17-negative CD4+ memory T cells. D. Observed statistical significance for differential gene expression between Th17-enriched and Th17-negative CD4+ T cells.
Table 1.
Immune-mediated-associated gene enrichment analysis.
Figure 3.
Preponderance of disease-associated transcripts upregulated in Th17 and in vitro differentiated Th1 cells.
Hierarchical clustering was performed on 195 transcripts with increased expression in Th17-enriched (red, increased expression relative to other cell types) compared to Th17-negative (blue, decreased expression) and differential expression P-values less than 1×10−09. Asterisks (*) designate the transcripts within immune-mediated disease loci. There are total 32 immune-mediated disease associated genes with names marked in the figure.
Figure 4.
Differential gene expression between CD4+ T cell subsets results from distinct molecular mechanisms.
Shown are the fractions of methylation at conserved CpG promoter sites estimated by mass spectrometry (N = 5) for A. IL23R, B. IL12RB2, C. IL17A and D. CCL20 promoters. Paired t-tests were used to test for differential methylation fractions between naïve vs. Th1 and Th17-negative vs. Th17-enriched were estimated by paired t-test; *P<0.05, **P<0.01, ***P<0.001. E. Average expression estimates from four individulas in both RNASeq and microarray show that Th17-specific transcripts IL17A and CCL20 have nearly zero expression in Th17-negative cells, corresponding to high promoter methylation levels (C–D). Gene expression was measured by FPKM (fragments of RNA per Kilobase of exon per Million fragments mapped) for RNASeq and log2 RMA normalized intensity for microarray.
Figure 5.
Abundantly expressed, novel IL23R isoform.
A. Sequence reads were mapped to the IL23R gene region in Th17-enriched and in vitro differentiated Th1 cells using Tophat v1.3.3. The intron 6 region was highly covered in both the Th17-enriched and Th1 cell subsets. The scale on the y-axis represents coverage (average number of reads that cover a particular base). B. Zoom in picture of extended coverage on exon 6 and intron 6. Blue and red bars correspond to sense- and anti-sense reads, respectively. The expanded inset demonstrates sequence reads mapping to an extended exon 6 resulting in a stop signal 9 codons downstream with contiguous 3′UTR sequence; in addition an independent transcript maps immediately centromeric to this. C. 3′ RACE (rapid amplification of cDNA ends) was performed, and 2000 and 700 base pair fragments were sequenced, confirming the exon contents designated. The 700 base pair transcript terminating in the intron 6 region would encode for a transcript terminating prior to the transmembrane domain in exon 9.