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

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.

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.

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.

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.

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.

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.

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.