Age and CD161 Expression Contribute to Inter-Individual Variation in Interleukin-23 Response in CD8+ Memory Human T Cells

<div><p>The interleukin-23 (IL-23) pathway plays a critical role in the pathogenesis of multiple chronic inflammatory disorders, however, inter-individual variability in IL-23-induced signal transduction in circulating human lymphocytes has not been well-defined. In this study, we observed marked, reproducible inter-individual differences in IL-23 responsiveness (measured by STAT3 phosphorylation) in peripheral blood CD8+CD45RO+ memory T and CD3+CD56+ NKT cells. Age, but not gender, was a significant (Pearson’s correlation coefficient, r = −0.37, p = 0.001) source of variability observed in CD8+CD45RO+ memory T cells, with IL-23 responsiveness gradually decreasing with increasing age. Relative to cells from individuals demonstrating low responsiveness to IL-23 stimulation, CD8+CD45RO+ memory T cells from individuals demonstrating high responsiveness to IL-23 stimulation showed increased gene expression for IL-23 receptor (IL-23R), RORC (RORγt) and CD161 (KLRB1), whereas RORA (RORα) and STAT3 expression were equivalent. Similar to CD4+ memory T cells, IL-23 responsiveness is confined to the CD161+ subset in CD8+CD45RO+ memory T cells, suggesting a similar CD161+ precursor as has been reported for CD4+ Th17 cells. We observed a very strong positive correlation between IL-23 responsiveness and the fraction of CD161+, CD8+CD45RO+ memory T cells (r = 0.80, p<0.001). Moreover, the fraction of CD161+, CD8+CD45RO+ memory T cells gradually decreases with aging (r = −0.34, p = 0.05). Our data define the inter-individual differences in IL-23 responsiveness in peripheral blood lymphocytes from the general population. Variable expression of CD161, IL-23R and RORC affects IL-23 responsiveness and contributes to the inter-individual susceptibility to IL-23-mediated defenses and inflammatory processes.</p> </div

The fraction of CD161+ subset is correlated with IL-23 responsiveness in CD8+CD45RO+ memory T cells and with age.

<p>(A) The fraction of the CD161+ subset in CD8+CD45RO+ memory T cells is positively correlated (n = 35, r = 0.80; p<0.001) with IL-23 responsiveness, measured by whole blood phospho-flow for IL-23-mediated pSTAT3 induction (log2). (B) Upper panel: FACS-sorted CD3+CD8+CD45RO+ CD161+ or CD161− T cells were stimulated with or without 100 ng/ml IL-23 for 15 minutes and pSTAT3 induction was assessed by phospho-flow. Representative flow cytometry plots are shown from one of the three individuals assessed. IL-23 responsive CD8+CD45RO+ T cells are confined to the CD161+ fraction. Lower panel: the percent IL-23R-expressing cells in CD8+CD161− or CD8+CD161+ cells are shown. Representative flow cytometry plots are shown from one of the two individuals assessed. (C) The fraction of the CD161+ subset in CD8+CD45RO+ memory T cells gradually declines with age (n = 35, r = −0.34, p = 0.05).</p

Selected results of differential gene expression analysis in CD8+CD45RO+ memory T cells from IL-23-responsive <i>vs.</i> -non-responsive individuals.

1<p>Genome-wide significance threshold (by Bonferroni correction): p = 0.05/11740 = 4.26×10⁻⁶.</p>2<p>Fold change is calculated as (mean expression level in IL-23-responsive subjects/mean expression level in IL-23-non-responsive subjects).</p

IL-23 responsiveness in CD8+CD45RO+ memory T cells is correlated with IL-23R, RORC and CD161 mRNA expression.

<p>Quantitative RT-PCR assays were performed in FACS-sorted CD8+CD45RO+ memory T cells from 15 individuals selected for having a range of IL-23 responsiveness, which were measured by whole blood phospho-flow assays for IL-23-mediated pSTAT3 induction (log2). Correlations were assessed between IL-23 responsiveness and (A) IL-23R, (B) RORC, (C) CD161, (D) RORA, and (E) STAT3 mRNA expression.</p

IL-23 stimulation of peripheral blood lymphocytes activates STAT proteins with inter-individual variability in IL-23 responsiveness.

<p>Peripheral whole blood was stimulated with 100ng/ml IL-23 for 15 minutes and IL-23-mediated pSTAT induction was assessed by phospho-flow assays for STAT1, 3, 4 and 5. (A) IL-23-mediated pSTAT induction in CD8+CD45RO+ memory T cells. Gray line and black lines represent pSTAT in un-stimulated and IL-23 stimulated samples, respectively. (B) IL-23-mediated pSTAT3 induction in CD8+ naïve (CD45RO-) and memory (CD45RO+) T cells, NK cells (CD3−CD56+), and NKT (CD3+CD56+) cells. (C) Representative individuals showing low (subject #1) or high (subject #2) IL-23 responsiveness.</p

Reproducibility of IL-23 responsiveness as assessed by pSTAT3 induction.

<p>Peripheral whole blood was stimulated with 100 ng/ml IL-23 for 15 minutes and pSTAT3 induction was assessed by phospho-flow assay. Repeated measures of IL-23-mediated pSTAT3 induction were performed for 25 randomly selected individuals, with the first and the second experiments separated by at least a three-week interval. IL-23 responsiveness was calculated as the log2 ratio of geometric mean fluorescence intensity (GMFI) of pSTAT3 in stimulated vs. unstimulated samples for (A) CD8+CD45RO+ memory T cells, and (B) CD3+CD56+ NKT cells. (C) IL-23-mediated pSTAT3 induction assessed by Western blot analysis in FACS sorted CD8+CD45RO+ memory T cells from individuals showing low (Group #1) or high (Group #2) IL-23-mediated pSTAT3 induction in phospho-flow assays.</p

Abundantly expressed, novel <i>IL23R</i> isoform.

<p>A. Sequence reads were mapped to the <i>IL23R</i> 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.</p

Differential gene expression between CD4+ T cell subsets results from distinct molecular mechanisms.

<p>Shown are the fractions of methylation at conserved CpG promoter sites estimated by mass spectrometry (N = 5) for A. <i>IL23R</i>, B. <i>IL12RB2</i>, C. <i>IL17A</i> and D. <i>CCL20</i> 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 <i>IL17A</i> and <i>CCL20</i> 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.</p

Preponderance of disease-associated transcripts upregulated in Th17 and in vitro differentiated Th1 cells.

<p>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.</p

CD4+ T cell subsets.

<p>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.</p