Four to six milliliters of blood was collected into EDTA-coated tubes from toddlers on visits when they had no fever, cold, flu, infections or other illnesses, or use of medications for illnesses 72 hours prior blood draw. Blood samples were passed over a LeukoLOCK™ filter (Ambion, Austin, TX, USA) to capture and stabilize leukocytes and immediately placed in a 20°C freezer. Total RNA was extracted following standard procedures and manufacturer’s instructions (Ambion, Austin, TX, USA). LeukoLOCK disks (Ambion Cat #1933) were freed from RNA-later and Tri-reagent (Ambion Cat #9738) was used to flush out the captured lymphocyte and lyse the cells. RNA was subsequently precipitated with ethanol and purified though washing and cartridge-based steps. The quality of mRNA samples was quantified by the RNA Integrity Number (RIN), values of 7.0 or greater were considered acceptable59 (link), and all processed RNA samples passed RIN quality control. Quantification of RNA was performed using Nanodrop (Thermo Scientific, Wilmington, DE, USA). Samples were prep in 96-well plates at the concentration of 25 ng/µl.