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Index of Online Supplemental Material for
J. Exp. Med. 10.1084/jem.20091669
Kisand et al.• Supplemental Material(PDF)
Article Figures & Data
Figures
- Figure 1.
Decreased IL-17F and IL-22 responses associate with CMC in APECED patients. (A) Secretion of IL-17A, IL-17F, and IL-22 by PBMCs from APECED patients without (CMC−) or with (CMC+) CMC and age-matched controls (Ctrl, n = 14 at 72-h time points) after stimulation with heat-killed C. albicans hyphae or SEB for 72 h was measured by ELISA. The patients are detailed in Table I. (B) SEB-stimulated PBMCs were stained intracellularly for IL-17A and IL-22 and FACS analyzed. Horizontal bars represent median values. A Kruskal-Wallis test was used to compare the group medians. This figure represents three independent experiments. Each symbol corresponds to a value obtained from an individual.
- Figure 2.
Disease-specificity of autoantibodies to IL-17A, IL-17F, and/ or IL-22. (A–C) Autoantibodies binding human IL-17A, IL-17F, and/ or IL-22 in sera from APECED or thymoma patients and disease or healthy controls tested by ELISA. (D and E) Comparisons of neutralizing and binding activity of anti–IL-17A and anti–IL-17F. (F) Because ELISA underestimates antibodies against IL-22, we used neutralizing assays for systematic comparisons. 12 sera were exhausted before exact titration of anti-IL-22 could be done. Red and black bars indicate group medians. SLE, systemic lupus erythematosus; RA, rheumatoid arthritis. Open circles represent patients T1 and T2 (Fig. 3 C and Table S2).
- Figure 3.
Autoimmunity to Th17-related cytokines is associated with CMC and cytokine productivity in vitro. (A) Associations between CMC and anti-cytokine autoantibodies in APECED patients. Red bars indicate group medians. (B) Representative FACS plots for intracellular IL-22 and IL-17A in a healthy subject (left) or in APECED patients with CMC and antibodies against IL-22 alone (middle) or against both IL-17A and IL-22 (right) after 6 h of SEB stimulation. (C) Production of IL-17A, IL-17F, and IL-22 by C. albicans– and SEB-stimulated (72 h) PBMCs from five random myasthenia gravis/thymoma patients was measured with ELISA. Patients T1 (X) and T2 (O) proved to be the only two with CMC. Median values for healthy controls are indicated with gray bars. (D) The concentration of secreted IL-17F (left) and IL-22 (right) after stimulating APECED patients’ PBMCs with SEB (for 72 and 18 h, respectively) correlates negatively with the respective neutralizing antibody titer. (E) Normal naive T cells were differentiated to Th17 cells with IL-1β, IL-6, IL-23, and IL-21 in the presence of sera (1:10) from either controls or APECED patients with antibodies to IL-22, IL-17F, and/or IL-17A. The IL-22– and IL-17A–producing cells were enumerated by flow cytometry. Data are representative of three independent experiments. Horizontal bars show median values.
Tables
Patient AIRE mutation Age CMC FACS sample Sample at 72 h Neutralizing antibody titer against: Binding antibodies against: a IFN-ω IL-17A IL-17F IL-22 IL-17A IL-17F IL-22 yr A c.[769C>T] + [769C>T]b 29 Yes No No 20,000 <32 38 9,000 0.12 3.11 0.17 Bc c.[21_43dup23] + [21_43dup23] 10 Yes Yes Yes >51,200 250,000 75 9,000 3.85 2.43 0.55 C c.[769C>T] + [769C>T] 17 Yes Yes Yes >51,200 <32 56 5,500 0.07 1.30 0.28 D c.[1064-1068dupCCCGG] + [1064-1068dupCCCGG] 18 Yes Yes Yes >51,200 <32 1,250 >525,000 0.83 3.64 1.81 E c.[769C>T] + [769C>T] 18 Yes No No >51,200 <32 350 160,000 0.70 3.52 1.01 F c.[769C>T] + [769C>T] 23 Yes No Yes 20,000 15,000 1,300 95,000 3.90 3.90 0.79 G c.[769C>T] + [769C>T] 60 Yes Yes Yes 12,800 <32 300 13,000 0.39 3.89 0.64 H c.[769C>T] + [967_979del13] 20 Yes No Yes 100,000 <32 5,500 4,500 2.98 3.90 0.25 I c.[967_979del13] + [1163_1164insA] 24 Yes No Yes 256,000 nd 9,200 3,300 0.09 3.90 0.56 J c.[879 + 1G>A] + [879 + 1G>A] 48 No Yes No 30,000 <32 <32 <32 0.30 0.29 0.18 Kc c.[967_979del13] + [967_979del13] 21 No Yes Yes >51,200 <32 <32 <32 0.17 0.40 0.24 L c.[769C>T] + [c.274C>T] 55 No No Yes 12,000 <32 <32 <32 0.04 0.03 0.16 M c.[967_979del13] + [c.274C>T] 29 No Yes Yes 200,000 <32 <32 930 0 0.03 0.21 Nd c. [682T>G] + [=] 40 No Yes Yes 9,000 <32 <32 <32 0.01 0.16 ND ↵a Positive is a value >0.4 for anti–IL-17A and anti–IL-17F and >0.2 for anti–IL-22.
↵b The mutation c.769C>T causes truncated AIRE protein p.R257X.
↵c PBMCs from patients B and K were tested repeatedly 6 mo apart. The results (unpublished data) were similar to the first stimulation.
↵d Dominant mutation p.G228W.
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