Targeted next generation sequencing of 589 genes identifies complementary responses in t-helper-like T regulatory cells

Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. However, they can also play a parallel role in the promotion of antitumor responses. Tregs can be classified as T helper (Th)-like Tregs that phenotypically resemble Th1/Th2/Th17/TH1-TH17 lineages based upon the expression of specific chemokine receptors and transcription factors. The distribution of these Treg subsets can provide mechanistic insights into disease processes, therefore we sought to characterise their immune transcriptome. Targeted RNA sequencing revealed that circulating Th2-like Tregs displayed higher viability and activation as well as suggesting specific disease pathways associated to sifferent T reg subtypes. Overall design: 2x105 FACS-sorted Th-like Tregs (TH1, TH2, TH17 abnd TH1-TH17) from 3 donors were activated with CD3/CD28 beads (ratio 1:4) for 72h. Cells were lysed in Trizol and RNA was isolated. RNA sequencing was performed using The Human Inflammation & Immunity Transcriptome RNA targeted panel (Qiagen). Samples were sequenced with Illumina MiSeq using MiSeq Reagent Kit v3 (150-cycle).