DOI: 10.5061/dryad.02v6wwq08
title: Data from: Gene editing to induce FOXP3 expression in human CD4+ T cells leads to a stable regulatory phenotype and function.

description: Thymic regulatory T cells (tTreg) are potent inhibitors of autoreactive
 immune responses and loss of tTreg function results in fatal autoimmune
 disease.  Defects in Treg number or function are also implicated
 in multiple autoimmune diseases, leading to growing interest in use of
 Treg as cell therapies to establish immune tolerance.  Because
 tTreg are present at low numbers in circulating blood and may be
 challenging to purify and expand, and also inherently defective in some
 subjects, we designed an alternative strategy to creating autologous
 Treg-like cells from bulk CD4+ T cells.  We utilized
 homology-directed-repair (HDR)-based gene-editing to enforce FOXP3
 expression.  Targeted insertion of a robust enhancer/promoter
 proximal to the first coding exon bypassed epigenetic silencing,
 permitting stable, high level endogenous FOXP3 expression. 
 HDR-edited T cells, edTreg, manifested a transcriptional program leading
 to sustained expression of canonical markers and suppressive activity of
 tTreg.  Both human and murine edTreg mediated immunosuppression
 in vivo in models of inflammatory disease.  Further, this
 engineering strategy permitted generation of antigen-specific edTreg with
 robust in vitro and in vivo functional activity.  Finally, edTreg
 could be enriched and expanded at scale using clinically-relevant
 methods.  Together, these finding suggest edTreg production may
 permit broad future clinical application.
description: RNA-seq was performed on tTreg, primary CD4+T cells (Teff) stably
 expressing a GFP-FOXP3 fusion protein using AAV6/CRISPR/Cas9 editing
 (=edTreg) , and non-edited CD4+ Teff cells from four healthy human
 donors.  tTreg and Teff had been previously sorted (CD4+CD25++CD127- and
 CD4+CD25-, respectively) and expanded with CD3/CD28 Dynabeads in media
 containing IL-2 and 0.1 µM rapamycin.  All cell cohorts were
 simultaneously re-activated with CD3/CD28 beads in IL-2 for 48 hours. 
 Beads were then removed, and these cells rested for another 48 hours in
 media containing IL-2 only.  Cells were sorted on the marker of interest
 directly into 4°C RNA lysis buffer on a FACSAria II (BD Biosciences), and
 RNA was purified using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing
 (Clontech).  cDNA libraries were then prepared using the SMARTseq V4 kit
 (Clontech).  1,000 cells from each group were directly sorted into lysis
 buffer.  Cells were re-sorted based on the following markers: Mock-edited
 cells were CD25-, edTreg were GFPhigh, and tTreg were CD25++CD127-. 
 Libraries were then prepared per manufacturer’s protocols, and RNAseq was
 performed on an Illumina HiSeq2500 platform.  RNA sequence reads were
 aligned to the human genome (hg19) using Tophat2.  Aligned reads mapping
 to the exons of a gene were quantitated with HTSeq-count.  DESeq2  was
 used to normalize the data using regularized-logarithm (rlog) and examine
 the relationship between samples.
description: File list: HONAKER_RNASEQ_Readme
 <br>
 Ctl1-activated-Treg_S330_L006_R1_001.fastq.gz<br>
 Ctl1-activated-eff_S329_L006_R1_001.fastq.gz<br>
 Ctl1-edit_S304_L006_R1_001.fastq.gz
 Ctl2-activated-Treg_S332_L006_R1_001.fastq.gz<br>
 Ctl2-activated-eff_S331_L006_R1_001.fastq.gz<br>
 Ctl2-edit_S327_L006_R1_001.fastq.gz <br>
 Ctl3-activated-Treg_S334_L006_R1_001.fastq.gz<br>
 Ctl3-activated-eff_S333_L006_R1_001.fastq.gz<br>
 Ctl3-edit_S306_L006_R1_001.fastq.gz
 Ctl4-activated-Treg_S336_L006_R1_001.fastq.gz<br>
 Ctl4-activated-eff_S335_L006_R1_001.fastq.gz<br>
 Ctl4-edit_S308_L006_R1_001.fastq.gz <br>
 Relationship between files:  <br> Ctl1, Ctl2, Ctl3, and Ctl4 were 4
 unique human peripheral blood donors;<br> activated Treg = activated
 tTreg;<br> edit = edTreg<br> activated eff = activated T
 effector cells