Mutations in STAT3 and IL12RB1 impair the development of human IL-17-producing T cells

Abstract

The cytokines controlling the development of human interleukin (IL) 17--producing T helper cells in vitro have been difficult to identify. We addressed the question of the development of human IL-17--producing T helper cells in vivo by quantifying the production and secretion of IL-17 by fresh T cells ex vivo, and by T cell blasts expanded in vitro from patients with particular genetic traits affecting transforming growth factor (TGF) beta, IL-1, IL-6, or IL-23 responses. Activating mutations in TGFB1, TGFBR1, and TGFBR2 (Camurati-Engelmann disease and Marfan-like syndromes) and loss-of-function mutations in IRAK4 and MYD88 (Mendelian predisposition to pyogenic bacterial infections) had no detectable impact. In contrast, dominant-negative mutations in STAT3 (autosomal-dominant hyperimmunoglobulin E syndrome) and, to a lesser extent, null mutations in IL12B and IL12RB1 (Mendelian susceptibility to mycobacterial diseases) impaired the development of IL-17--producing T cells. These data suggest that IL-12Rbeta1- and STAT-3--dependent signals play a key role in the differentiation and/or expansion of human IL-17-producing T cell populations in vivo.

Figure 1.

Identification of IL-17–producing T cells ex vivo. (A) Flow cytometry analysis of CD3 and IL-17 in nonadherent PBMCs activated with PMA-ionomycin as a representative control, an IRAK-4–deficient patient (P4), an IL-12Rβ1–deficient patient (P17), and a STAT-3–deficient patient (P36; Table S1, available at http://www.jem.org/cgi/content/full/jem.20080321/DC1). The percentage indicated in the gate is that of IL-17– and CD3-positive cells. (B) Percentage of CD3-positive cells that were also IL-17–positive, as determined by flow cytometry of nonadherent PBMCs activated with PMA-ionomycin. Each symbol represents a value from an individual control (black circles) or patient (red circles). Horizontal bars represent medians. The p-values for Wilcoxon tests between controls (n = 49) and patients with mutations in IRAK4 or MYD88 (n = 9), IL12B or IL12RB1 (n = 17), TGFB1 or TGFBR2 (n = 7), and STAT3 (n = 16) are indicated.

Figure 2.

Identification of IL-17–expressing T cell blasts expanded in vitro. Intracellular production of IL-17 in T cell blasts activated with PMA-ionomycin for controls (black circles) and patients (red circles), as assessed by flow cytometry. The cells were cultured in different stimulation conditions: OKT3 only (A), or OKT3 with IL-23 (B), IL-1β (C), or IL-23, IL-1β, TGF-β, and IL-6 (D). Each symbol represents a value for an individual control or patient. Horizontal bars represent medians. In controls, stimulation with IL-23 and IL-1β had a significant effect with respect to medium alone (P < 0.05). The p-values for Wilcoxon tests between each patient group and the control group are indicated. In B and D, the patients circled in blue carry IL12B mutations and cannot produce IL-12 and IL-23, but can respond to both cytokines. The p-value of the IL12B-IL12RB1 group was therefore calculated only with IL-12Rβ1–deficient patients (*).

Figure 3.

IL-17 secretion by T cell blasts expanded in vitro. Secretion of IL-17 by T cell blasts from controls (black circles) and patients (red circles), as measured by ELISA. Open circles represent values in the absence of stimulation, and closed circles correspond to values obtained after stimulation with PMA-ionomycin. Different stimulation conditions are shown: OKT3 only (A), or OKT3 with IL-23 (B), IL-1β (C), or IL-23, IL-1β, TGF-β, and IL-6 (D). Each symbol corresponds to a value obtained from an individual. Horizontal bars represent medians. The p-values for Wilcoxon tests between each patient group and the control group, either unstimulated or stimulated with PMA-ionomycin, are indicated. In B and D, patients circled in blue carry IL12B mutations and cannot produce IL-12 and IL-23, but can respond to both cytokines. The p-values of the IL12B-IL12RB1 group were therefore calculated only with IL-12Rβ1–deficient patients (*).