A novel form of complete IL-12/IL-23 receptor β1 deficiency with cell surface-expressed nonfunctional receptors by Claire Fieschi, Marita Bosticardo, Ludovic de Beaucoudrey, Stéphanie Boisson-Dupuis, Jacqueline Feinberg, Orchidée Filipe Santos, Jacinta Bustamante, Jacov Levy, Fabio Candotti, and Jean-Laurent Casanova Blood Volume 104(7):2095-2101 October 1, 2004 ©2004 by American Society of Hematology

A large in-frame deletion in IL12RB1. A large in-frame deletion in IL12RB1. (A) Schematic representation of the wild-type IL-12Rβ1 chain containing 17 coding exons (Arabic numerals) encoding 662 amino acids, with a peptide leader sequence (L), extracellular domain (exons 2 to 13, EC), transmembrane domain (exon 14, TM), and an intracellular, cytoplasmic domain (exons 15 to 17, IC). The mutation found in P is also indicated (700 + 362_1619-944 del). The mature IL-12Rβ1 chain contains 5 fibronectin III (FNIII) domains shown in the bottom row in light gray. (B) Schematic representation of the mutant protein, lacking the sequences encoded by 6 of the exons (8 to 13) in the wild-type gene. The mutant protein contains 356 amino acids and only the first 2 FNIII domains of the extracellular domain but has intact transmembrane and intracellular domains. In the family tree, the patient is indicated by an arrow. Claire Fieschi et al. Blood 2004;104:2095-2101 ©2004 by American Society of Hematology

IL-12Rβ1 chain detected at the cell surface by FACS analysis. IL-12Rβ1 chain detected at the cell surface by FACS analysis. PHA-T-cell blasts from a positive control (C+), the patient (P), and a negative control (C-) were stained with various purified mouse monoclonal antibodies (24E6, B101, B103, 12RB44), rat mAb (2B10), or matched isotype control, followed by biotinylated matched Ab and phycoerythrin-conjugated streptavidin. IL-12Rβ1 clone 69310, directly conjugated to R-PE, was compared with a matched conjugated isotype control. Specific signals are represented as plain lines; matched isotype controls are represented as dotted lines. Claire Fieschi et al. Blood 2004;104:2095-2101 ©2004 by American Society of Hematology

Lack of phosphorylated STAT4 upon IL-12 stimulation, as shown by FACS analysis. Lack of phosphorylated STAT4 upon IL-12 stimulation, as shown by FACS analysis. PHA-T-cell blasts from a positive control (C+), a negative control (C-), and the patient (P) were left unstimulated (plain line) or were stimulated (dotted line) with IFN-α (105 U/mL) (left) for 30 minutes or with IL-12 (100 ng/mL) (right) for 15 minutes. Cells were fixed by PFA and methanol, permeabilized with saponin, and stained with a phospho-STAT4 rabbit polyclonal Ab (Zymed) (or matched isotype control), followed by goat anti-rabbit Alexa Fluor 488 (Molecular Probes). Claire Fieschi et al. Blood 2004;104:2095-2101 ©2004 by American Society of Hematology

Lack of IFN-γ production in response to IL-12 and IL-23. Lack of IFN-γ production in response to IL-12 and IL-23. PHA-T-cell blasts from a positive control (C+), a negative control (C-), and the patient (P) were plated in 24-well plates and were left unstimulated (NS) or were stimulated with increasing concentrations of IL-12 for 48 hours (A) or IL-23 for 72 hours (B). Supernatants were harvested, and IFN-γ was quantified by ELISA. Claire Fieschi et al. Blood 2004;104:2095-2101 ©2004 by American Society of Hematology

Lack of cytokine binding to the surface of PHA-T-cell blasts. Lack of cytokine binding to the surface of PHA-T-cell blasts. PHA-T-cell blasts from a positive control (C+), a negative control (C-), and the patient (P) were incubated without (dotted line) or with (plain line) IL-12p70 or IL-23 for 30 minutes at 4°C. The PHA-T-cell blasts were then incubated with a purified mouse anti-IL-12 p40 antibody, followed by a biotinylated anti-mouse antibody, and antibody binding was detected by incubation with PE-conjugated streptavidin. Claire Fieschi et al. Blood 2004;104:2095-2101 ©2004 by American Society of Hematology

Correction of the patient's IL-12Rβ1 defect by retroviral-mediated gene tranfer. Correction of the patient's IL-12Rβ1 defect by retroviral-mediated gene tranfer. (A) PHA-T-cell blasts from the patient (P), a negative control (C-), MND-IL-12Rβ1-transduced PHA-T-cell blasts from the patient (Ptd), or the negative control (C-td) were stained with anti-IL-12Rβ1 mAb (24E6 or 2B10, plain line) or matched isotype control (dotted line). (B) IFN-γ production in response to IL-12. PHA-T-cell blasts from the patient (P), a negative control (C-), and MND-IL-12Rβ1-transduced PHA-T-cell blasts from the patient (Ptd) or the negative control (C-td) were plated in 24-well plates and were either left unstimulated (NS) or were stimulated with increasing concentrations of IL-12 for 48 hours. Supernatants were harvested, and IFN-γ was quantified by ELISA. Claire Fieschi et al. Blood 2004;104:2095-2101 ©2004 by American Society of Hematology