Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

PBMCs were isolated by density centrifugation through Ficoll-Hypaque PLUS (GE Healthcare Life Sciences). Pan T cells were isolated by negative selection using Pan T Cell Isolation kit (Miltenyi Biotec) to ≥98% purity. Pan T cells were stimulated with anti-CD2/CD3/CD28 coated beads from the T Cell Activation/Expansion kit (Miltenyi Biotec), at a bead to cell ratio of 1:1. T cells were cultured in RPMI medium supplemented with 10% FBS (Gibco), 100 U/ml recombinant human IL-2 (Aldesleukin, Prometheus), 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Gibco), and 55 µM 2-ME (Sigma-Aldrich). Where indicated, previously activated cycling T cells were treated with 100 IU/ml recombinant human IFN-alpha-2b (Intron A; Merck) for 20 h to induce MDA5 expression.