Inflammatory disease protective R381Q IL23 receptor polymorphism results in decreased primary CD4+ and CD8+ human T-cell functional responses

Ritu Sarin; Xingxin Wu; Clara Abraham

doi:10.1073/pnas.1017854108

Edited by Warren Leonard, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board April 27, 2011 (received for review November 29, 2010)

Abstract

The SNP (c.1142G > A;p.R381Q) in the IL-23 receptor (IL23R) confers protection from multiple inflammatory diseases, representing one of the most significant human genetic polymorphisms in autoimmunity. We, therefore, sought to define the functional consequences of this clinically significant variant. We find that CD4+CD45RO+ and CD8+ T cells from healthy R381Q IL23R carriers show decreased IL-23–dependent IL-17 and IL-22 production relative to WT IL23R individuals. This was associated with a lower percentage of circulating Th17 and Tc17 cells. Furthermore, CD8+ T cells from R381Q IL23R individuals showed decreased IL-23–dependent expansion and signal transducer and activator of transcription 3 (STAT3) activation compared with WT CD8+ T cells. Importantly, cells transfected with the IL23R glutamine variant show decreased IL-23–mediated signaling compared with the IL23R arginine allele. Our results show that the R381Q IL23R variant leads to selective, potentially desirable, loss of function alterations in primary human CD4+ and CD8+ T cells, resulting in highly significant protection against autoimmunity.

The IL-23/Th17 pathway is critical for optimal microbial defenses (1, 2); however, disregulation of this pathway can lead to inflammation (3 –9). The SNP (c.1142G > A; p.R381Q) in IL-23 receptor (IL23R) confers protection from inflammatory diseases, including ankylosing spondylitis, psoriasis, and inflammatory bowel disease (10 –12); it represents one of the most significant human genetic polymorphisms in autoimmunity. On binding to the IL23R complex, IL-23 mediates its effects by signaling through the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and NF-κB pathways (13, 14). IL-23 modulates responses in multiple cell populations, including differentiation and maintenance of CD4+ Th17 (6, 15 –17) and CD8+ Tc17 cells (18 –21).

We considered that the disease-protective R381Q IL23R may result in a loss of function, leading to decreased IL-23/Th17 pathway cytokine production, or a gain of function, leading to enhanced microbial clearance. We find that CD4+CD45RO+ and CD8+ T cells from healthy R381Q IL23R carriers show decreased IL-23–dependent IL-17 and IL-22 production relative to WT IL23R individuals. This was associated with a lower percent of circulating Th17 and Tc17 cells. Furthermore, R381Q CD8+ T cells showed decreased IL-23– and STAT3-dependent expansion and STAT3 activation compared with WT cells. Our results indicate that the protective R381Q IL23R variant leads to selective loss of function in primary human CD4+ and CD8+ T cells.

Results

Memory CD4+ T Cells from R381Q IL23R Individuals Show Decreased IL-23–Mediated Th17 Cytokine Production on in Vitro Stimulation.

To define the role of the disease-protective R381Q IL23R polymorphism, we genotyped 650 healthy individuals and identified 48 individuals carrying the minor glutamine allele, consistent with prior estimates of a 7% allele frequency (11). WT individuals are controls not carrying the R381Q IL23R allele. Because of increased IL23R expression on memory CD4+ T cells (13, 22), we used CD4+CD45RO+ T cells to examine the consequences of R381Q IL23R on Th17 cells. We first assessed cytokine responses from anti-CD3/anti-CD28–activated R381Q or WT IL23R CD4+CD45RO+ T cells for 4 d in the presence or absence of IL-23. Activation in the presence of IL-23 led to increased IL-17 and IL-22 secretion from both WT and R381Q IL23R CD4+CD45RO+ T cells (Fig. 1A) within the range of prior reports (22). However, concentrations of IL-23–mediated IL-17 and IL-22 secretion were significantly decreased in R381Q compared with WT IL23R CD4+CD4RO+ T cells (Fig. 1A). In contrast, secreted IFN-γ concentrations were equivalent between the two groups. Consistent with cytokine secretion results, IL-23–stimulated R381Q CD4+CD45RO+ T cells showed a decreased percentage of IL-17– and IL-22–producing cells (Fig. 1 B and C).

Fig. 1.

Decreased IL-23–mediated Th17 cytokine production on in vitro stimulation of CD4+CD45RO+ T cells from R381Q IL23R individuals. (A–C) Purified WT and R381Q IL23R CD4+CD45RO+ T cells were activated for 4 d with anti-CD3/CD28 mAbs in the presence or absence of IL-23 (10 ng/mL). (A) IL-17, IL-22, and IFN-γ were measured in supernatants and represented as mean + SEM for WT (n = 21) and R381Q (n = 13) individuals. (B) Representative flow plots depicting IL-23–stimulated cytokine-producing CD4+CD45RO+ T cells. (C) Mean percentage of cytokine-producing CD4+CD45RO+ T cells + SEM for WT (n = 12–15) and R381Q (n = 9–10) individuals. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

R381Q IL23R Individuals Have Decreased Percentages of Circulating CD4+ Th17 Cells Relative to WT IL23R Individuals.

Decreased IL-23–mediated IL-17 and IL-22 production by in vitro stimulated R381Q CD4+CD45RO+ T cells might be caused by decreased IL-23–dependent cell expansion or cytokine induction in vitro and/or altered circulating Th17 populations. Importantly, IL-23 did not increase expansion of either WT or R381Q IL23R CD4+CD45RO+ T cells relative to cells activated in the absence of IL-23 under these short-term conditions (Fig. S1). We next questioned if R381Q individuals contained fewer circulating Th17 CD4+ T cells. Studies have identified that CCR6+ and CD161+ human CD4+ T cells are enriched for IL-17–producing cells (23, 24). However, neither the percentage of circulating CD4+CD45RO+ T cells (Fig. S2A) nor the percentage of CCR6+, CD161+, or CCR6+CD161+CD4+CD45RO+ T cells (Fig. S2 B and C) was significantly different between WT and R381Q IL23R individuals.

Examining CCR6+CD161+ T cells may not be adequately sensitive to detect differences in IL-17–producing T cells in R381Q and WT IL23R individuals, because only ∼20–30% of CCR6+CD161+CD4+ T cells produce IL-17 (24). Therefore, we directly examined the percentages of IL-17– and IL-22–producing CD4+ T cells ex vivo on stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin. We detected a decreased percentage of circulating IL-17– and IL-22–producing T cells from both peripheral total CD4+ (Fig. S2 D and E) and fractionated CD4+CD45RO+ T cells (Fig. 2 A and B) in R381Q relative to WT IL23R individuals. In contrast, the percentage of circulating IFN-γ–producing CD4+ T cells (Fig. S2 D and E) and CD4+CD45RO+ T cells (Fig. 2 A and B) was not significantly different between the two groups. Consistent with the decreased circulating CD4+CD45RO+ Th17-lineage cells in R381Q individuals, we observed a decreased percentage of IL23R-expressing cells within the CD4+CD45RO+ T cells of R381Q carriers (Fig. 2 C and D). Of note, surface protein expression on CD4+CD45RO+ T cells was not significantly different between R381Q and WT IL23R individuals (Fig. 2 C and E), although we cannot exclude small differences in surface expression.

Fig. 2.

Decreased ex vivo circulating CD4+CD45RO+ Th17 cells in R381Q IL23R individuals. Purified CD4+CD45RO+ T cells from WT and R381Q IL23R individuals were stimulated ex vivo with PMA/ionomycin for 5 h to assess for circulating cytokine producing cells on day 0 (A and B). (A) Representative flow plots and (B) graphs showing the mean percent + SEM of circulating cytokine-producing CD4+CD45RO+ T cells for WT (n = 12) and R381Q (n = 9–10) healthy donors. Peripheral blood mononuclear cells (PBMCs) were stained for IL23R and gated on CD4+CD45RO+CD45RA− T cells. Shown are (C) representative flow plots and graphs depicting (D) the percentage of IL23R-expresssing cells, with the solid line representing the mean, and (E) IL23R surface expression as assessed by mean fluorescence intensity (MFI) on IL23R+ CD4+CD45RO+ T cells from WT (n = 27) and R381Q (n = 13) individuals. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

CD8+ T Cells from R381Q IL23R Individuals Show Decreased IL-23–Mediated Tc17 Cytokine Production and Expansion on in Vitro Stimulation.

We next assessed CD8+ T cells from R381Q IL23R individuals. Of note is that fewer CD8+ T cells ex vivo (e.g., 0.2%) or on differentiation in vitro produce IL-17 compared with CD4+ T cells (20). Similar to CD4+ T cells and compared with WT IL23R CD8+ T cells, R381Q CD8+ T cells showed significantly decreased percentages of IL-17– and IL-22–producing cells on activation in the presence of IL-23 (Fig. 3 A and B); in contrast, the percentage of IFN-γ–producing CD8+ T cells was equivalent (Fig. 3 A and B).

Fig. 3.

Decreased IL-23–mediated Tc17 cytokine production and expansion in vitro in CD8+ T cells from R381Q IL23R individuals. CD8+ T cells were purified from WT and R381Q IL23R individuals, activated for 4 d as in Fig.1, and assessed for production of IL-17, IL-22, and IFN-γ by intracellular cytokine staining (A and B). (A) Representative flow plots with isotype controls in the presence of IL-23 and (B) graphs showing mean percent cytokine-producing CD8+ T cells + SEM for WT (n = 14–16) and R381Q (n = 7) individuals. (C) Graph showing fold increase in live cell numbers on day 5 on activation with anti-CD3/CD28 mAbs in the presence or absence of IL-23 relative to cell numbers at initial plating. Data represent mean + SEM for WT (n = 11) and R381Q (n = 8) individuals. The dotted line represents a value of 1. *P ≤ 0.05; **P ≤ 0.01.

Given the decreased percentages of IL-23–dependent IL-17– and IL-22–producing CD8+ T cells from R381Q individuals on in vitro stimulation, we questioned if IL-23 contributes to human CD8+ T-cell expansion, and if so, whether R381Q CD8+ T cells have decreased IL-23–mediated expansion. Purified WT and R381Q IL23R CD8+ T cells were stimulated in the presence or absence of IL-23 for 5 d. In contrast to CD4+ T cells, IL-23 enhanced expansion of anti-CD3/anti-CD28–activated CD8+ T cells from WT IL23R individuals by approximately twofold relative to non–IL-23–treated cells. Importantly, IL-23 did not enhance expansion of R381Q CD8+ T cells (Fig. 3C). Therefore, decreased IL-23–mediated expansion of R381Q CD8+ T cells may be one mechanism contributing to decreased IL-23–mediated production of CD8+ Tc17 lineage cytokines during in vitro stimulation.

R381Q IL23R Individuals Have Decreased Percentages of Circulating CD8+ Tc17 Cells Relative to WT IL23R Individuals.

We next questioned if the decreased Tc17 lineage cytokine production on in vitro stimulation might also be because of decreased circulating Tc17 cells in R381Q individuals. CCR6+CD161− and CCR6+CD161+CD8+ T cells were not different in R381Q compared with WT IL23R individuals (Fig. S3). However, direct ex vivo circulating IL-17– and IL-22–producing CD8+ T cells were significantly decreased in R381Q compared with WT IL23R individuals (Fig. 4 A and B). In contrast, percentages of circulating IFN-γ–producing CD8+ T cells were not significantly different (Fig. 4 A and B). There was a decreased percentage of CD8+ T cells from R381Q individuals expressing IL23R relative to WT individuals (Fig. 4 C and D). However, no significant difference in per cell IL23R surface expression among the small fraction of IL23R-expressing cells in CD8+ T cells was observed (Fig. 4 C and E).

Fig. 4.

Decreased ex vivo circulating CD8+ Tc17 cells in R381Q IL23R individuals. PBMCs from WT and R381Q IL23R individuals were either PMA/ionomycin-stimulated and stained for intracellular cytokines (A and B) or stained unstimulated for IL23R (C–E) and gated on CD8+ T cells. (A) Representative flow plots with isotype controls and (B) graphs showing the mean percent + SEM of circulating cytokine-producing CD8+ T cells for WT (n = 22–23) and R381Q (n = 13–14) individuals on day 0. (C) Representative flow plots and graphs showing (D) the percentage of IL23R+ cells gated on CD8+ T cells, with the solid line representing the mean, and (E) IL23R surface expression as assessed by MFI on IL23+ CD8+ T cells from WT (n = 27) and R381Q (n = 14) IL23R individuals. **P ≤ 0.01; ^†P ≤ 0.0001; ^††P ≤ 0.00001.

CD8+ T Cells from R381Q IL23R Individuals Show Decreased IL-23–Mediated STAT3 Activation, and STAT3 Is Required for IL-23–Mediated CD8+ T-Cell Expansion.

IL-23 activates STAT3 in both human and mouse cells (13, 25, 26). STAT3 is required for IL-17 and IL-22 secretion and likely contributes to IL-17–producing T-cell expansion and/or differentiation (25, 27 –29). We, therefore, investigated STAT3 activation downstream of R381Q and WT IL23R in CD8+ T cells. Of note, we found that the STAT3 rs744166 T risk allele (30) was equivalent between the two IL23R cohorts in our studies. R381Q and WT CD8+ T cells were first activated under Tc17 skewing conditions. Under these conditions of supplemental cytokines, cell expansion, percentages of IL23R-expressing cells (Fig. S4A), and IL23R surface expression (Fig. S4B) are equivalent between R381Q and WT IL23R CD8+ T cells. Equal numbers of either R381Q or WT CD8+ T cells were then stimulated with IL-23 for 30 min. Relative to WT cells, CD8+ T cells from R381Q IL23R individuals showed reduced STAT3 phosphorylation on IL-23 stimulation (Fig. 5 A and B and Fig. S5). In contrast, STAT3 phosphorylation in R381Q CD8+ T cells in response to IL-21 and IL-10 was intact (Fig. S5 A and B), indicating that the loss of STAT3 activation is specific to IL-23 stimulation.

Fig. 5.

R381Q IL23R CD8+ T cells show decreased IL-23–mediated STAT3 activation. CD8+ T cells from R381Q and WT IL23R individuals were stimulated under Tc17-differentiating conditions to equally expand T cells, and 2 wk later, equal cell numbers were treated for 30 min with or without IL-23 (10 ng/mL). Up-regulation of pSTAT3 was determined by immunoblotting. (A) Representative immunoblot and (B) graph quantitating the fold pSTAT3 up-regulation after equalizing for total STAT3 and normalizing to non–IL-23–treated cells. Data represent the mean + SEM for WT (n = 9) and R381Q (n = 7) individuals. (C) WT CD8+ T cells were stimulated as in Fig. 3C in the presence or absence of IL-23 (10 ng/mL) and in the presence or absence of the STAT3 inhibitor peptide SIP (50 μM) for 5 d. Cell numbers were determined, and fold expansion of CD8+ T cells relative to original plated numbers was calculated. Data represent the mean + SEM for WT (n = 21) individuals. ***P ≤ 0.001; ^††P ≤ 0.00001.

Given that R381Q CD8+ T cells show decreased IL-23–mediated cell expansion (Fig. 3C) and STAT3 activation (Fig. 5 A and B) compared with WT CD8+ T cells, we questioned if IL-23–mediated STAT3 activation contributes to the CD8+ T-cell expansion. The role of STAT3 in CD8+ T-cell expansion has not been clearly addressed. WT IL23R CD8+ T cells were activated in the presence or absence of IL-23 as per Fig. 3C and in the presence or absence of a STAT3 inhibitor peptide (SIP). Significantly, the IL-23–mediated increase in CD8+ T-cell expansion was lost on STAT3 inhibition (Fig. 5C), showing that the lack of IL-23–induced STAT3 activation in R381Q CD8+ T cells likely contributes to the loss of IL-23–mediated expansion in CD8+ T cells from these individuals.

R381Q IL23R Results in a Direct Loss of IL-23–Mediated Signaling Relative to WT IL23R.

The decreased IL-23–mediated cell expansion (Fig. 3C) and Tc17 cytokine production (Fig. 3 A and B) in CD8+ T cells from R381Q IL23R individuals might be because of the decreased percentage of IL23R-expressing cells and/or decreased signaling through R381Q IL23R. To further address these two possibilities, we used two approaches. First, we examined the equally expanded WT and R381Q Tc17 cells (as per Fig. 5) with equivalent percentages of IL23R-expressing cells for expansion and cytokine production after reactivation in the presence of IL-23. The fold expansion of R381Q IL23R CD8+ T cells remained decreased compared with WT cells (Fig. S6A). In contrast to WT CD8+ T cells, R381Q IL23R CD8+ T-cell expansion was unaffected by the presence of SIP (Fig. S6A), consistent with the decreased STAT3 activation in these T cells. With reactivation, decreased IL-17–producing cells in R381Q CD8+ T cells were observed, whereas IFN-γ production was equivalent (Fig. S6 B and C).

In a second approach, we examined expression and signaling of WT or R381Q-IL23R on cotransfection with IL12Rβ1 into cells to allow for expression of the functional IL23R complex. Surface IL23R and IL12Rβ1 expression were equivalent when comparing transfection of WT and R381Q-IL23R along with IL12Rβ1 into HEK293 cells; these receptors are not expressed in untransfected HEK293 cells (Fig. 6 A and B). NF-κB is required for IL-23–mediated IL-17 production in mice (14), and we observed that NF-κB luciferase activity was decreased in R381Q compared with WT-IL23R transfected cells on IL-23 stimulation (Fig. S7). However, we were unable to observe pSTAT3 expression on IL-23 stimulation in these same cells, likely because of a lack of the required upstream JAK proteins. We, therefore, examined other cells lines for induction of pSTAT3 expression on IL-23 stimulation. We found that, although IL-23 stimulation of HeLa cells was unable to induce pSTAT3 on transfection of the empty vector, pSTAT3 expression was induced on transfection of both WT IL23R and IL12Rβ1 (Fig. 6 C and D). Consistent with our findings in primary T cells, STAT3 phosphorylation was decreased in R381Q relative to WT IL23R-transfected cells on IL-23 stimulation (Fig. 6 C and D). Moreover, we observed decreased STAT3 luciferase activity 24 h after IL-23 stimulation in R381Q IL23R relative to WT IL23R-transfected cells (Fig. 6E), showing that the altered STAT3 phosphorylation ultimately leads to decreased STAT3-mediated transcriptional activation. Taken together, these results indicate, through studies in both primary cells and transfected cells, that the R381Q IL23R variant results in decreased signaling relative to WT IL23R.

Fig. 6.

Cells transfected with R381Q IL23R show decreased STAT3 activation compared with WT IL23R transfected cells. HEK293 cells were transiently transfected in duplicate or triplicate with either WT or R381Q IL23R cDNA along with IL-12R β1 cDNA, and 24 h after transfection, cells were assessed for IL23R and IL12Rβ1 surface expression by flow cytometry. (A) Representative histogram of IL23R (Upper) and IL12Rβ1 (Lower) surface expression (the dark gray-shaded area indicates isotype staining, the light gray-shaded area indicates control DNA transfected cells, the dark solid line indicates WT IL23R cDNA-transfected cells, and the dark dotted line indicates R381Q IL23R cDNA-transfected cells) and (B) graph quantitating IL23R and IL12Rβ1 surface expression as measured by MFI for five and three independent experiments, respectively. HeLa cells were transfected with either WT or R381Q IL23R cDNA along with IL12Rβ1 cDNA (C and D) and STAT3-luciferase and Renilla (E) or empty vectors, where appropriate; 24 h after transfection, cells were incubated in the absence or presence of IL-23 (10 ng/mL) for 30 min (C and D) or 24 h (E). (C) Shown is a representative immunoblot for pSTAT3 and total STAT3 expression and (D) a graph quantitating pSTAT3 expression normalized for total STAT3. Data represent the mean + SEM for samples run in duplicate. Data are representative of three independent experiments. (E) Graph showing the fold STAT3-luciferase reporter activity of IL-23–treated or untreated cells normalized to Renilla luciferase activity in WT or R381Q IL23R transfected cells run in triplicate. Data are representative of three independent experiments. NT, no treatment; EV, empty vector. **P < 0.01.

Discussion

In this study, we report, using primary human T cells and transfected cells, functional alterations associated with the R381Q IL23R polymorphism, which provides mechanistic insight into its protection against inflammatory diseases. That the essential role of IL-23 and Th17 cell subsets in mediating mucosal defense and inflammation was only recently defined reflects the challenges of studying these cell subsets, in part because of their very modest fraction in circulating lymphocytes. We have shown impaired IL-23–mediated STAT3 signaling (Fig. 5) and expansion (Fig. 3C) in CD8+ T cells heterozygous for the R381Q polymorphism as well as decreased IL-23–mediated induction of IL-17 and IL-22 in CD4+ (Fig. 1 A–C) and CD8+ (Fig. 3 A and B) T cells. The latter finding likely reflects both the effects of impaired IL-23 signaling as well as decreased percentages of Th17 (Fig. 2 A and B) and Tc17 subsets (Fig. 4 A and B) observed in R381Q heterozygotes compared with WT individuals.

We show through multiple lines of evidence that there is a decrease in function of the R381Q IL23R variant. One study using retrovirally transduced IL23R variants in human T-cell blasts identified no difference between the R381Q and WT forms of IL23R in IFN-γ secretion and STAT3 phosphorylation; this study was unable to detect IL-17 secretion (31). We similarly did not observe differences in IFN-γ secretion between WT and R381Q IL23R T cells, because the decreased cytokine secretion was selective to IL-17 and IL-22. The inability of the prior study to detect STAT3 differences between the mutant and WT IL23R variants could be because of a number of reasons, including the examination of retrovirally transduced T-cell blasts rather than isolated primary CD8+ T cells expressing endogenous WT or R381Q IL23R. The use of primary cells from WT and R381Q IL23R individuals in our study provides particular value in defining the consistent and highly significant findings presently reported.

Relative to WT IL23R, we observe decreased STAT3 activation in primary CD8+ T cells from R381Q IL23R carriers and cells transfected with R381Q IL23R. JAK2 associates constitutively with IL23R, and STAT3 is recruited to the complex on IL23R stimulation (13). JAK proteins generally associate through their 4.1, ezrin, radixin, moesin (FERM) domain with membrane-proximal regions of cytokine receptors (32). R381 is in the proximal IL23R cytoplasmic tail at the fifth amino acid from the putative transmembrane region, and it is conserved between species. Therefore, the R381Q IL23R variant may affect constitutive association of JAK2 with IL23R, with effects on subsequent STAT3 recruitment, phosphorylation, and transcriptional activation.

Interestingly, we observe shared and distinct consequences of IL-23 in CD4+ and CD8+ T cells. T-cell activation in the presence of IL-23 results in increased IL-17 and IL-22 production in both CD4+ and CD8+ T cells. However, in contrast to the IL-23–mediated enhanced expansion in CD8+ T cells and loss of this expansion in R381Q IL23R CD8+ T cells, memory CD4+ T cells did not show a role for IL-23 in short-term in vitro expansion (Fig. S1). This is consistent with previously reported short-term expansion of human CD4+ Th17 clones (33) and CD4+ T cells in mice (16). It remains possible that other conditions may induce IL-23–driven expansion, such as longer-term CD4+ memory T-cell expansion or naïve CD4+ T-cell expansion under Th17 differentiation conditions. The decreased IL-17 production in CD4+ and CD8+ T cells from individuals carrying the disease-protective R381Q IL23R variant relative to WT IL23R carriers is consistent with the general contribution of IL-17 to inflammation (2). In contrast, IL-22 has more complex functions with significant effects on epithelial cells. Although IL-22 contributes to epidermal hyperplasia and inflammation in skin, it down-regulates inflammation in the intestine, in part through maintaining epithelial barrier integrity (2). As a result, under specific situations, decreased IL-22 expression may lead to adverseconsequences.

The decreased IL23R-expressing Th17 and Tc17 subsets in R381Q healthy carriers reflect the cumulative effects of altered IL-23 function in vivo. Thus far, there have been no reports that R381Q carriers are at increased risk for infectious complications. It remains possible that, under certain conditions (e.g., high levels of Th17-differentiating cytokines or specific microbial infections), R381Q IL23R carriers may be able to mount normal Th17 responses. The present findings of decreased IL23R function associated with the R381Q IL23R protective allele highlight a potentially desirable selective functional alteration that balances adequate defense with protection against excessive inflammation.

Materials and Methods

Patient Recruitment and Genotyping.

Informed consent from healthy donors between ages 21 and 55 y was obtained per Institutional Review Board protocol. Genotyping by Sequenom platform (Sequenom) was used to assess R381Q IL23R (rs11209026;c1142G > A).

Purification of CD4+ Memory and CD8+ T-Cell Subsets.

CD4+CD45RO+ (>95% pure) and CD8+ T cells (>95% pure) were obtained using two consecutive MACS LS columns. PBMCs were first depleted of B cells and monocytes using an anti-CD19 and anti-CD14 antibody mixture (Miltenyi Biotech) combined with anti-CD45RA and anti-CD8 microbeads (for memory CD4+ T cells) or anti-CD4+ microbeads (for total CD8+ T cells). CD4+CD45RO+ or CD8+ T cells were then positively selected with either anti-CD4 or anti-CD8 microbeads, respectively.

Antibodies.

Flow cytometry antibodies included anti–CD3-FITC, anti–CD4-PE-cy5/APC, anti–CD8-APC or -PE, anti–CD45RO-FITC/PE-cy5, anti–CD45RA-FITC/APC (Ebiosciences), anti–CCR6-PE, anti–CD161-PE-cy5, anti–IFNγ-PE, anti–IL-17-Alexafluor 647, anti–IL12Rβ1-PE, isotypes mIgG-PE/Alexafluor 647 (BD), anti–IL23R-PE, anti–IL-22-PE, and isotype mIgG2B (R&D Systems). Anti-CD3, anti-CD28, anti–IL-4, and anti–IFN-γ were obtained from R&D Systems.

Surface and Intracellular Cytokine Staining.

For IL23R surface staining, 2 × 10⁶ PBMCs were blocked with 10% human AB serum and then stained with IL23R-PE or an isotype control. Cells were acquired on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (TreeStar). For intracellular cytokine staining, cells were incubated with PMA, ionomycin, and brefeldin A (Sigma) for 5 h. Cells were stained with anti-CD3/CD8 mAbs followed by 4% paraformaldehyde fixation and 0.5% saponin permeabilization and then, were stained with cytokine mAbs. Because PMA/ionomycin stimulation down-regulates the CD4+ marker, CD3+CD8− cells were gated and analyzed to identify CD4+ T cells (34).

T-Cell Cultures.

CD4+CD45RO+ or CD8+ T cells were either seeded at 0.025 × 10⁶ cells (for T-cell expansion) or 0.05 × 10⁶ cells (for cytokine production) per well on 96-well plates coated with anti-CD3 (5 μg/mL) and anti-CD28 (1 μg/mL) and were cultured in RPMI 1640 supplemented with 10% FBS at 37 °C in the absence or presence of IL-23 (10 ng/mL; R&D Systems). Secreted IL-17 and IL-22 (R&D Systems) and IFN-γ (BD Biosciences) were measured by ELISA. STAT3 inhibitor peptide (EMD Chemicals) was used for STAT3 inhibition.

Immunoblotting.

Purified CD8+ T cells (0.25 × 10⁶) were cultured in RPMI supplemented with IL-1β (10 ng/mL), IL-6 (40 ng/mL), IL-23 (10 ng/mL), anti–IFN-γ (5 μg/mL), and anti–IL-4 (5 μg/mL) on anti-CD3/CD28 mAbs-coated plates. R381Q and WT CD8+ T cells activated with the addition Tc17 skewing cytokines expanded equally; 2 wk later, cells were cultured with or without IL-23 for 30 min at 37 °C and examined for pSTAT3 (Cell Signaling) by immunoblot.

HEK293 and HeLa Cell Transfections.

A glutamine at position 381 in WT IL23R was generated through site-directed mutagenesis (QuikChange XL Site-Directed Mutagenesis kit; Agilent Technologies) of IL23R cDNA (Origene Technologies), and the mutation was confirmed by sequencing. HEK293 or HeLa cells were transiently transfected in duplicate or triplicate as indicated with either WT or R381Q IL23R cDNA along with IL-12R β1cDNA (GeneCopoeia), and in some cases, STAT3 luciferase combined with Renilla (Qiagen) or control vectors where appropriate; 24 h after transfection, cells were incubated in the absence or presence of IL-23 (10 ng) for the indicated times and assessed for luciferase activity (Promega) or pSTAT3 expression by immunoblot.

Statistical Analysis.

Paired t test was used to analyze cells in the presence and absence of IL-23. Mann–Whitney U test was used to compare WT IL-23R and R381Q IL-23R–expressing individuals.

Acknowledgments

We thank the blood donors, John Karpinski for technical assistance, and Maria-Luisa Alegre, Judy Cho, Joseph Craft, Fred Gorelick, and Ben Turk for critical reading of the manuscript and/or helpful advice. This work was supported by Grants from the National Institutes of Health: R01DK077905-02S1, DKP30-34989, and U19-AI082713.

Footnotes

↵¹To whom correspondence should be addressed. E-mail: clara.abraham{at}yale.edu.

Author contributions: C.A. designed research; R.S., X.W., and C.A. performed research; R.S., X.W., and C.A. analyzed data; and R.S. and C.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. W.L. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1017854108/-/DCSupplemental.

References

↵
1. McKenzie BS,
2. Kastelein RA,
3. Cua DJ
(2006) Understanding the IL-23-IL-17 immune pathway. Trends Immunol 27:17–23.
OpenUrl CrossRef PubMed
↵
1. Abraham C,
2. Cho JH
(2009) IL-23 and autoimmunity: New insights into the pathogenesis of inflammatory bowel disease. Annu Rev Med 60:97–110.
OpenUrl CrossRef PubMed
↵
1. Cua DJ,
2. et al.
(2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744–748.
OpenUrl CrossRef PubMed
↵
1. Park H,
2. et al.
(2005) A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6:1133–1141.
OpenUrl CrossRef PubMed
↵
1. Yen D,
2. et al.
(2006) IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest 116:1310–1316.
OpenUrl CrossRef PubMed
↵
1. Awasthi A,
2. et al.
(2009) Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells. J Immunol 182:5904–5908.
OpenUrl Abstract/FREE Full Text
↵
1. Hue S,
2. et al.
(2006) Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J Exp Med 203:2473–2483.
OpenUrl Abstract/FREE Full Text
↵
1. Ahern PP,
2. Izcue A,
3. Maloy KJ,
4. Powrie F
(2008) The interleukin-23 axis in intestinal inflammation. Immunol Rev 226:147–159.
OpenUrl CrossRef PubMed
↵
1. Elson CO,
2. et al.
(2007) Monoclonal anti-interleukin 23 reverses active colitis in a T cell-mediated model in mice. Gastroenterology 132:2359–2370.
OpenUrl CrossRef PubMed
↵
1. Cargill M,
2. et al.
(2007) A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet 80:273–290.
OpenUrl CrossRef PubMed
↵
1. Duerr RH,
2. et al.
(2006) A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 314:1461–1463.
OpenUrl Abstract/FREE Full Text
↵
1. Wellcome Trust Case Control Consortium
(2007) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447:661–678.
OpenUrl CrossRef PubMed
↵
1. Parham C,
2. et al.
(2002) A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. J Immunol 168:5699–5708.
OpenUrl Abstract/FREE Full Text
↵
1. Cho ML,
2. et al.
(2006) STAT3 and NF-kappaB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice. J Immunol 176:5652–5661.
OpenUrl Abstract/FREE Full Text
↵
1. Wilson NJ,
2. et al.
(2007) Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol 8:950–957.
OpenUrl CrossRef PubMed
↵
1. McGeachy MJ,
2. et al.
(2009) The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo. Nat Immunol 10:314–324.
OpenUrl CrossRef PubMed
↵
1. Volpe E,
2. et al.
(2008) A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat Immunol 9:650–657.
OpenUrl CrossRef PubMed
↵
1. Ciric B,
2. El-behi M,
3. Cabrera R,
4. Zhang GX,
5. Rostami A
(2009) IL-23 drives pathogenic IL-17-producing CD8+ T cells. J Immunol 182:5296–5305.
OpenUrl Abstract/FREE Full Text
↵
1. Intlekofer AM,
2. et al.
(2008) Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin. Science 321:408–411.
OpenUrl Abstract/FREE Full Text
↵
1. Kondo T,
2. Takata H,
3. Matsuki F,
4. Takiguchi M
(2009) Cutting edge: Phenotypic characterization and differentiation of human CD8+ T cells producing IL-17. J Immunol 182:1794–1798.
OpenUrl Abstract/FREE Full Text
↵
1. Curtis MM,
2. Way SS,
3. Wilson CB
(2009) IL-23 promotes the production of IL-17 by antigen-specific CD8 T cells in the absence of IL-12 and type-I interferons. J Immunol 183:381–387.
OpenUrl Abstract/FREE Full Text
↵
1. Chen Z,
2. Tato CM,
3. Muul L,
4. Laurence A,
5. O'Shea JJ
(2007) Distinct regulation of interleukin-17 in human T helper lymphocytes. Arthritis Rheum 56:2936–2946.
OpenUrl CrossRef PubMed
↵
1. Acosta-Rodriguez EV,
2. et al.
(2007) Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol 8:639–646.
OpenUrl CrossRef PubMed
↵
1. Cosmi L,
2. et al.
(2008) Human interleukin 17-producing cells originate from a CD161+CD4+ T cell precursor. J Exp Med 205:1903–1916.
OpenUrl Abstract/FREE Full Text
↵
1. de Beaucoudrey L,
2. et al.
(2008) Mutations in STAT3 and IL12RB1 impair the development of human IL-17-producing T cells. J Exp Med 205:1543–1550.
OpenUrl Abstract/FREE Full Text
↵
1. Oppmann B,
2. et al.
(2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715–725.
OpenUrl CrossRef PubMed
↵
1. Zhou L,
2. et al.
(2007) IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol 8:967–974.
OpenUrl CrossRef PubMed
↵
1. Huber M,
2. et al.
(2009) A Th17-like developmental process leads to CD8(+) Tc17 cells with reduced cytotoxic activity. Eur J Immunol 39:1716–1725.
OpenUrl CrossRef PubMed
↵
1. Nurieva R,
2. et al.
(2007) Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature 448:480–483.
OpenUrl CrossRef PubMed
↵
1. Barrett JC,
2. et al.
(2008) Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nat Genet 40:955–962.
OpenUrl CrossRef PubMed
↵
1. de Paus RA,
2. van de Wetering D,
3. van Dissel JT,
4. van de Vosse E
(2008) IL-23 and IL-12 responses in activated human T cells retrovirally transduced with IL-23 receptor variants. Mol Immunol 45:3889–3895.
OpenUrl CrossRef PubMed
↵
1. Schindler C,
2. Plumlee C
(2008) Inteferons pen the JAK-STAT pathway. Semin Cell Dev Biol 19:311–318.
OpenUrl CrossRef PubMed
↵
1. Annunziato F,
2. et al.
(2007) Phenotypic and functional features of human Th17 cells. J Exp Med 204:1849–1861.
OpenUrl Abstract/FREE Full Text
↵
1. Streeck H,
2. et al.
(2008) Rapid ex vivo isolation and long-term culture of human Th17 cells. J Immunol Methods 333:115–125.
OpenUrl CrossRef PubMed

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Proceedings of the National Academy of Sciences: 108 (23)

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[1] ↵
McKenzie BS,
Kastelein RA,
Cua DJ
(2006) Understanding the IL-23-IL-17 immune pathway. Trends Immunol 27:17–23.
OpenUrl CrossRef PubMed

[2] McKenzie BS,

[3] Kastelein RA,

[4] Cua DJ

[5] ↵
Abraham C,
Cho JH
(2009) IL-23 and autoimmunity: New insights into the pathogenesis of inflammatory bowel disease. Annu Rev Med 60:97–110.
OpenUrl CrossRef PubMed

[6] Abraham C,

[7] Cho JH

[8] ↵
Cua DJ,
et al.
(2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744–748.
OpenUrl CrossRef PubMed

[9] Cua DJ,

[10] et al.

[11] ↵
Park H,
et al.
(2005) A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6:1133–1141.
OpenUrl CrossRef PubMed

[12] Park H,

[13] et al.

[14] ↵
Yen D,
et al.
(2006) IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest 116:1310–1316.
OpenUrl CrossRef PubMed

[15] Yen D,

[16] et al.

[17] ↵
Awasthi A,
et al.
(2009) Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells. J Immunol 182:5904–5908.
OpenUrl Abstract/FREE Full Text

[18] Awasthi A,

[19] et al.

[20] ↵
Hue S,
et al.
(2006) Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J Exp Med 203:2473–2483.
OpenUrl Abstract/FREE Full Text

[21] Hue S,

[22] et al.

[23] ↵
Ahern PP,
Izcue A,
Maloy KJ,
Powrie F
(2008) The interleukin-23 axis in intestinal inflammation. Immunol Rev 226:147–159.
OpenUrl CrossRef PubMed

[24] Ahern PP,

[25] Izcue A,

[26] Maloy KJ,

[27] Powrie F

[28] ↵
Elson CO,
et al.
(2007) Monoclonal anti-interleukin 23 reverses active colitis in a T cell-mediated model in mice. Gastroenterology 132:2359–2370.
OpenUrl CrossRef PubMed

[29] Elson CO,

[30] et al.

[31] ↵
Cargill M,
et al.
(2007) A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet 80:273–290.
OpenUrl CrossRef PubMed

[32] Cargill M,

[33] et al.

[34] ↵
Duerr RH,
et al.
(2006) A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 314:1461–1463.
OpenUrl Abstract/FREE Full Text

[35] Duerr RH,

[36] et al.

[37] ↵
Wellcome Trust Case Control Consortium
(2007) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447:661–678.
OpenUrl CrossRef PubMed

[38] Wellcome Trust Case Control Consortium

[39] ↵
Parham C,
et al.
(2002) A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. J Immunol 168:5699–5708.
OpenUrl Abstract/FREE Full Text

[40] Parham C,

[41] et al.

[42] ↵
Cho ML,
et al.
(2006) STAT3 and NF-kappaB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice. J Immunol 176:5652–5661.
OpenUrl Abstract/FREE Full Text

[43] Cho ML,

[44] et al.

[45] ↵
Wilson NJ,
et al.
(2007) Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol 8:950–957.
OpenUrl CrossRef PubMed

[46] Wilson NJ,

[47] et al.

[48] ↵
McGeachy MJ,
et al.
(2009) The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo. Nat Immunol 10:314–324.
OpenUrl CrossRef PubMed

[49] McGeachy MJ,

[50] et al.

[51] ↵
Volpe E,
et al.
(2008) A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat Immunol 9:650–657.
OpenUrl CrossRef PubMed

[52] Volpe E,

[53] et al.

[54] ↵
Ciric B,
El-behi M,
Cabrera R,
Zhang GX,
Rostami A
(2009) IL-23 drives pathogenic IL-17-producing CD8+ T cells. J Immunol 182:5296–5305.
OpenUrl Abstract/FREE Full Text

[55] Ciric B,

[56] El-behi M,

[57] Cabrera R,

[58] Zhang GX,

[59] Rostami A

[60] ↵
Intlekofer AM,
et al.
(2008) Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin. Science 321:408–411.
OpenUrl Abstract/FREE Full Text

[61] Intlekofer AM,

[62] et al.

[63] ↵
Kondo T,
Takata H,
Matsuki F,
Takiguchi M
(2009) Cutting edge: Phenotypic characterization and differentiation of human CD8+ T cells producing IL-17. J Immunol 182:1794–1798.
OpenUrl Abstract/FREE Full Text

[64] Kondo T,

[65] Takata H,

[66] Matsuki F,

[67] Takiguchi M

[68] ↵
Curtis MM,
Way SS,
Wilson CB
(2009) IL-23 promotes the production of IL-17 by antigen-specific CD8 T cells in the absence of IL-12 and type-I interferons. J Immunol 183:381–387.
OpenUrl Abstract/FREE Full Text

[69] Curtis MM,

[70] Way SS,

[71] Wilson CB

[72] ↵
Chen Z,
Tato CM,
Muul L,
Laurence A,
O'Shea JJ
(2007) Distinct regulation of interleukin-17 in human T helper lymphocytes. Arthritis Rheum 56:2936–2946.
OpenUrl CrossRef PubMed

[73] Chen Z,

[74] Tato CM,

[75] Muul L,

[76] Laurence A,

[77] O'Shea JJ

[78] ↵
Acosta-Rodriguez EV,
et al.
(2007) Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol 8:639–646.
OpenUrl CrossRef PubMed

[79] Acosta-Rodriguez EV,

[80] et al.

[81] ↵
Cosmi L,
et al.
(2008) Human interleukin 17-producing cells originate from a CD161+CD4+ T cell precursor. J Exp Med 205:1903–1916.
OpenUrl Abstract/FREE Full Text

[82] Cosmi L,

[83] et al.

[84] ↵
de Beaucoudrey L,
et al.
(2008) Mutations in STAT3 and IL12RB1 impair the development of human IL-17-producing T cells. J Exp Med 205:1543–1550.
OpenUrl Abstract/FREE Full Text

[85] de Beaucoudrey L,

[86] et al.

[87] ↵
Oppmann B,
et al.
(2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715–725.
OpenUrl CrossRef PubMed

[88] Oppmann B,

[89] et al.

[90] ↵
Zhou L,
et al.
(2007) IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol 8:967–974.
OpenUrl CrossRef PubMed

[91] Zhou L,

[92] et al.

[93] ↵
Huber M,
et al.
(2009) A Th17-like developmental process leads to CD8(+) Tc17 cells with reduced cytotoxic activity. Eur J Immunol 39:1716–1725.
OpenUrl CrossRef PubMed

[94] Huber M,

[95] et al.

[96] ↵
Nurieva R,
et al.
(2007) Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature 448:480–483.
OpenUrl CrossRef PubMed

[97] Nurieva R,

[98] et al.

[99] ↵
Barrett JC,
et al.
(2008) Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nat Genet 40:955–962.
OpenUrl CrossRef PubMed

[100] Barrett JC,

[101] et al.

[102] ↵
de Paus RA,
van de Wetering D,
van Dissel JT,
van de Vosse E
(2008) IL-23 and IL-12 responses in activated human T cells retrovirally transduced with IL-23 receptor variants. Mol Immunol 45:3889–3895.
OpenUrl CrossRef PubMed

[103] de Paus RA,

[104] van de Wetering D,

[105] van Dissel JT,

[106] van de Vosse E

[107] ↵
Schindler C,
Plumlee C
(2008) Inteferons pen the JAK-STAT pathway. Semin Cell Dev Biol 19:311–318.
OpenUrl CrossRef PubMed

[108] Schindler C,

[109] Plumlee C

[110] ↵
Annunziato F,
et al.
(2007) Phenotypic and functional features of human Th17 cells. J Exp Med 204:1849–1861.
OpenUrl Abstract/FREE Full Text

[111] Annunziato F,

[112] et al.

[113] ↵
Streeck H,
et al.
(2008) Rapid ex vivo isolation and long-term culture of human Th17 cells. J Immunol Methods 333:115–125.
OpenUrl CrossRef PubMed

[114] Streeck H,

[115] et al.

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