Human Regulatory T cells from Umbilical Cord Blood Display Increased Repertoire Diversity and Lineage Stability Relative to Adult Peripheral Blood

Microarray used to detail bulk transcriptomic differences between sorted CD4+CD25+CD127lo/- Treg and CD4+CD25-CD127+ Tconv from adult peripheral blood (APB) and cord blood (CB) after a 14 day expansion period. CB and APB Tregs and Tconv were FACS sorted as CD4+CD25hiCD127lo and CD4+CD127+, respectively, on a BD FACS Aria III. Both subsets were incubated with anti-CD3 anti-CD28 coated microbeads in the presence of exogenous IL-2 and expanded for 14 days following protocol 1 as described in Seay et al, 2017. 3E5 CB Tregs, CB Tconv, APB Tregs, and APB Tconv were lysed in DNA/RNA lysis buffer (Zymo ResearchR) and stored at -80°C. RNA extraction was achieved using ZR-Duet™ DNA/RNA MiniPrep (Zymo Research, Catalog No. D7001), per the manufacturer’s instructions. Quality assessment of RNA was achieved by Experion™ Automated Electrophoresis System (BIO-RAD) using Experion RNA High Sensitivity Reagents and Experion Standard Sensitivity RNA chips following the manufacturer protocol. Only samples with a minimum RNA concentration of 10 ng/μL and RNA Quality Index (RQI) greater than or equal to 9.4 were used. Briefly, mRNA was reverse transcribed and amplified, and resulting cDNA was fragmented and labeled using Encore Biotin Module (Nugen) and subsequently, hybridized onto GeneChip Human Transcriptome Array 2.0 (Thermo Scientific), following the manufacturer’s procedures.