A male Caucasian infant was born after an uneventful 37-week pregnancy, as the first child to nonconsanguineous parents. His neonatal course was uncomplicated, and he was fed initially with breast milk alone and then in combination with artificial milk. He received standard immunizations at birth and at 2 months without complications. He did not receive any live virus vaccines. At the age of 2 months, he started suffering from gastrointestinal problems with feeding intolerance, frequent episodes of diarrhea, and episodes of fever with leukocytosis and neutrophilia. By that time, failure to thrive was evident.
At 7 months of age he was referred for evaluation and admitted to the intensive care unit with respiratory impairment; extensive interstitial lung disease was evident by a chest computerized tomography scan, which also identified a normal-sized thymus. Bronchoscopy revealed parainfluenza virus and Pneumocystis carinii, which was successfully treated with trimethoprim-sulfa-methoxazole. In his first months of life he also suffered from recurrent episodes of bacteremia. Testing excluded HIV infection. A skin biopsy from the antecubital region revealed an absence of sweat glands, and the results of two sweat tests supported the diagnosis of anhidrosis. The patient underwent an allogeneic cord blood transplant and expired in the peritransplant period due to gram-negative sepsis.
The patient, family members, and unaffected normal controls were studied at the National Jewish Medical and Research Center and the Clinical Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIAID/NIH; protocol 89-I-006). Immunologically normal adult individuals served as controls. The Institutional Review Board of the NIAID approved the open protocol, and informed consent was obtained from the parents before the enrollment in the study.
Lymphocyte subpopulations were studied by flow cytometry on at least three occasions prior to transplant with the whole-blood lysis technique and analyzed with a FACScan (Becton Dickinson, San Jose, CA) with CellQuest software. The monoclonal antibodies used included anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD16, anti-CD56, anti-CD45RA, anti-CD45RO, anti-CD20, anti-CD5, anti-IgG, anti-IgM, anti-IgA, and anti-CD27 (all from Becton Dickinson). Irrelevant antibodies to the different subclasses were used to ascertain background staining. To calculate the absolute number of each subpopulation, the percentage was multiplied by the absolute count of peripheral blood lymphocytes as determined by Coulter counter (Celdyne, San Jose, CA) followed by a differential cell leukocyte count of a blood sample that had been obtained simultaneously.
Cell Preparation and Culture Conditions
Peripheral blood mononuclear cells (PBMCs) were obtained from patients and healthy adult volunteers by centrifugation of heparinized blood over Ficoll-Hypaque density gradient lymphocyte separation medium (Pharmacia Biotech, Piscataway, NJ), with standard techniques. To measure interleukin IL-12 production, 2 × 106 PBMCs were cultured in RPMI 1640 complete medium (1 ml) for 36 hr. The following reagents were also used: human interferon (IFN)-γ (1,000 U/ml; Peprotech, Rocky Hill, NJ), SAC (Pansorbin cells, 1:10,000 w/v, Calbiochem, La Jolla, CA), LPS from E. coli 01127:B8 (1 μg/ml; Sigma, St. Louis, MO), OspA (a gift of Alan Sher, Bethesda, MD), and CD40L trimer (2.5 μg/ml; a gift of Immunex Corp., Seattle, WA). For anti-CD3 stimulation, OKT3 antibody (gift of Ortho Biotech, Somerset, NJ) was first dissolved in carbonate buffer at a concentration of 10 μg/ml and aliquotted into 24-well plates at 250 μl/well. After overnight incubation at 4°C, the plates were washed twice in sterile PBS. Cell suspensions were then added. The cytokine IL-12 was used (10 μg/ml; R&D Systems, Minneapolis, MN). After 36 hr, supernatants were removed. IFN-γ, IL-12, and TNF-α concentrations were determined by specific ELISA (R&D Systems), according to the manufacturer’s instructions.
DNA and RNA were extracted from lymphocytes by standard methods. A NEMO
gene study was performed as previously described [Jain et al., 2001
]. For IKBA
gene, exons 1–6 were amplified by PCR from genomic DNA with primers flanking each exon as described [Courtois et al., 2003
]. The PCR products were purified (Qiagen, Valencia, CA) and cycle-sequenced at both the 5′ and the 3′ ends with the dye-terminator dideoxy nucleotides. Sequencing results were compared to the reference sequence GenBank NM_020529.1 and positions numbered as position 1 being the A of the first ATG initiation codon.
Western Blot Studies
Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines cultured in RPMI with 10% fetal calf serum (FCS) were stimulated with CD40L trimer (2.5 μg/ml) in the presence of cycloheximide (CHX, 50 μg/ml; Sigma), an inhibitor of protein translation, to prevent de novo IκBα protein synthesis. Cell lysates were prepared by resuspending cell pellet in 100 mM NaCl, 50 mM Tris-Cl at pH 8.0, 0.5% NP-40, 50 mM NaF, 30 mM sodium pyrophosphate, 1 mM Na3VO4, 0.6 % diisopropyl fluorophosphates (Sigma), and 1 × complete TM protease inhibitor mixture (Boehringer Mannheim, Indianapolis, IN) for 15 min on ice followed by centrifugation at 38,000 rpm for 30 min. The supernatants were collected as cell lysates and the protein concentration determined by the Bio-Rad protein assay method (Bio-Rad, Hercules, CA) with bovine albumin as a standard. Samples were then applied to SDS-PAGE followed by transfer onto nitrocellulose membranes. The membranes were probed with either anti-IκBα N-terminal specific, or anti-IκBα C-terminal specific antibody (catalog numbers SC203 and SC271; Santa Cruz Biotech, Santa Cruz, CA). This was followed by the addition of horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham, Piscataway, NJ). Films were developed by the enhanced chemiluminescence (ECL) method (Amersham). For protein loading control, membranes were stripped and reblotted with anti-β-actin specific antibody (Sigma).
T Cell Proliferation Assay
PBMCs were suspended in complete medium (RPMI containing 1% penicillin/streptomycin with 10% FCS) at 3 × 106 cells/ml. In triplicate wells, the PBMC cultures were either unstimulated or stimulated with phytohemagglutinin (PHA) diluted 1:100 (In-vitrogen, Carlsbad, CA) or tetanus toxoid (1:400 concentration; Connaught Laboratories, Philadelphia, PA), or Conclavin A (Con-A) 5 μg/ml (Invitrogen), or Pokweed mitogen (PWM) 1 μg/ml (Sigma-Aldrich, St. Louis, MO). Tetanus toxoid cultures were pulsed after 4 days and all other culture conditions were pulsed at 2 days with 1 μCi of [3H]thymidine per well (ICN Biomedicals, Costa Mesa, CA) and incubated an additional 18 hr before harvesting onto printed filter mats (Wallac/Perkins Elmer, Waltham, MA) using a 96-well cell harvester (TOMTEC, Orange, CA). Incorporation of 3H on filter mats was measured as counts per minute (cpm) by a BetaPlate reader (Wallac/Perkins Elmer).
Luciferase Reporter Gene Assay
Human embryonic kidney 293 (HEK293) cells were seeded into 24-well plates (1 × 105 cells/well). Following an 8-hr incubation, the cells were transfected with 1.1 μg of a luciferase expressing reporter plasmid under an NF-κB promoter (pNF-κB-Luc) (BD Biosciences) with 0.2 μg β-galactosidase control vector as a transfection control to normalize luciferase activity. A total of 2.2 μg of either an IκBα–wild-type-expressing plasmid (pCMV-IKBA-WT), an IκBα–mutant-expressing plasmid (pCMV-IKBA-40G>T), or empty vector (pCMV-empty). Transfections were carried out using a Calcium Phosphate Transfection Kit (Invitrogen). Approximately 36 hr after transfection, the cells were stimulated with TNF-α (10 ng/ml) for 6 hr. The cells were then harvested and luciferase activity was measured with a Bright-GloTM luciferase assay system (Promega, Madison, WI). The presented data represents one of three independent transfection experiments. Transfections with pCMV-IKBA-WT and pCMV-IKBA-40G>T were carried out in triplet. Luciferase activity of each sample was normalized to an internal reference standard of β-galactosidase activity.