Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells

Ng MSF, Roth TL, Mendoza VF, Marson A, Burt TD
Source: Science
Publication Date: (2019)
Issue: 4(41): eaav5947
Research Area:
Basic Research
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® 96-well Systems

Cultivation: 4 biological replicates of sorted adult or fetal naïve T cells were stimulated in U-bottomed 96 wells with 5 µl of Immunocult Human T cell Activator (CD3/CD28/CD2 tetramers, STEMCELL Technologies) in 200 µl of culture media (RPMI-1640 supplemented with 10% HI-FBS (heat inactivated fetal bovine serum), 300 mg/L L-glutamine, 10 U/mL penicillin, and 10 µg/mL streptomycin, 10 mM HEPES, 1X MEM-NEAA, ß-mercaptoethanol). Exogenous IL-2 (10 ng/ml, Peprotech) and TGF-ß (50 ng/ml, Peprotech) were added according to the relevant experimental setup. 

CRISPR-Cas9 editing of the Helios locus: 

Cas9 gRNA ribonucleoproteins (RNPs) were assembled before ex-periments by assembling the gRNAs through incubating 160 µM CRISPR RNA (crRNA) with 160 µM trans-activating crRNA (tracrRNA) (Dharmacon) at a 1:1 ratio for 30 min at 37°C to a final concentration of 80 µM.Assembled gRNAs were then subsequently incubated with 40 µM Cas9-nuclear localization sequence (NLS) (QB3 MacroLab, University of California, Berkeley) at a 1:1 ratio for 15 min at 37°C for a final concentration of 20 µM RNPs. Nucleo-fection was performed using the Amaxa P3 Primary Cell 96-well Nucleofector Kit and 4D-Nucleofector (Lonza). A total of 1 × 10^5 sorted fetal naïve T cells per well were stimulated overnight in 96-well plates precoated with anti-CD3 antibodies (1 µg/ml; HIT3a, BD Biosciences) and supplemented with soluble anti-CD28 antibodies (2 µg/ml; CD28.2, BD Biosciences) and IL-2 (10 ng/ml; PeproTech). Stimulated fetal naïve T cells were washed with PBS and resuspended in 20 µl of P3 solution. Five microliters of the final 20 µM RNP solution was added, along with 1 µl of 100 µM homology directed repair template (HDRT) solution. Cells were gently mixed and transferred to the 96-well shuttle device. Cells were electroporated using program EH-115 on the Amaxa 4D-Nucleofector (Lonza).


T cell receptor (TCR) stimulation and cytokine cues drive the differentiation of CD4+ naïve T cells into effector T cell populations with distinct proinflammatory or regulatory functions. Unlike adult naïve T cells, human fetal naïve CD4+ T cells preferentially differentiate into FOXP3+ regulatory T (Treg) cells upon TCR activation independent of exogenous cytokine signaling. This cell-intrinsic predisposition for Treg differentiation is implicated in the generation of tolerance in utero; however, the underlying mechanisms remain largely unknown. Here, we identify epigenetic and transcriptional programs shared between fetal naïve T and committed Treg cells that are inactive in adult naïve T cells and show that fetal-derived induced Treg (iTreg) cells retain this transcriptional program. We show that a subset of Treg-specific enhancers is accessible in fetal naïve T cells, including two active superenhancers at Helios Helios is expressed in fetal naïve T cells but not in adult naïve T cells, and fetal iTreg cells maintain Helios expression. CRISPR-Cas9 ablation of Helios in fetal naïve T cells impaired their differentiation into iTreg cells upon TCR stimulation, reduced expression of immunosuppressive genes in fetal iTreg cells such as IL10, and increased expression of proinflammatory genes including IFNG Consequently, Helios knockout fetal iTreg cells had reduced IL-10 and increased IFN-? cytokine production. Together, our results reveal important roles for Helios in enhancing preferential fetal Treg differentiation and fine-tuning eventual Treg function. The Treg-biased programs identified within fetal naïve T cells could potentially be used to engineer enhanced iTreg populations for adoptive cellular therapies.