Human blood and tissue

Adult peripheral human blood samples were obtained from the Australian Red Cross Blood Service under agreement number 13-04VIC-07. Young human peripheral blood samples and matching thymus were obtained from the Royal Children’s Hospital (RCH; Victoria, Australia). These thymic and matched peripheral blood data were derived from eight donors between the ages of 2 months and 4 years old.

Experiments were conducted in accordance with the University of Melbourne Human Research and Ethics committee guidelines (reference numbers 1035100 and 1443540) and RCH Human Research Ethics Committee Approval (reference number 24131 G). Blood mononuclear cells were isolated by Ficoll-Paque Plus density gradient centrifugation (GE Healthcare). Donor thymi were cut into small pieces and passed through a 70-μm cell strainer into ice-cold RPMI 1640 medium containing 2 mM EDTA before being washed into phosphate-buffered saline + 2% fetal calf serum [fluorescence-activated cell sorting (FACS) buffer].