Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity

Abstract

SummaryTreg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we targeted the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-knockout Tregs upregulated cytokine expression, effects on suppressive capacity manifested slowly and primarily in memory Tregs. Moreover, FOXP3-knockout Tregs retained their characteristic phenotype and had few changes in their DNA methylation landscape, with FOXP3 maintaining methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.

Helios is a marker, not a driver, of human Treg stability

“…This possibility is supported by the finding that human fetal naive T‐cells deficient in Helios are unable to differentiate into Tregs in vitro in the absence of TGF‐β [27]. Similarly, our investigation into the role of FOXP3 in mature human Tregs revealed that continuous expression of this master transcription factor is dispensable for maintenance of human Treg identity [21]. Overall, high Helios expression marks stable Tregs whereas Helios mid identifies destabilized Tregs, but Helios expression per se does not have a direct role in maintaining this lineage‐committed state.…”

“…Treg expansion with CRISPR editing was performed as described previously (Supporting information Fig. S1B) [21]. Cells were preactivated for 5 days with IL-2 and either 1:1 aAPCs or 25 μL/mL CD3/CD28/CD2 tetramer (ImmunoCult Human CD3/CD28/CD2 T Cell Activator, STEMCELL Technologies).…”

“…S1D and S3 were transfected with Cas9. During optimization of Treg expansion with CRISPR/Cas9 editing, we found superior Treg expansion with aAPC-based preactivation relative to CD3/CD28/CD2 tetramers; in both scenarios, cells were restimulated once or twice with aAPCs as above [21]. Comparing the two preactivation methods, we found that aAPC-preactivated nTregs, mTregs, and Tconvs displayed similar FOXP3 and Helios kinetics (not shown), and nTregs exhibited comparable in vitro suppressive function at day 20 (Supporting information Fig.…”

“…S1A) were stimulated every 5-7 days following a protocol compatible with efficient CRISPR/Cas9 editing (Supporting information Fig. S1B) [21].…”

Regulatory T Cells-Related Genes Are under DNA Methylation Influence

“…In reference to retroviral CAR transfection, lung epithelial-directed Tregs were able to diminish the immune response in a murine airway hyper-reactivity model [169]. The CRISPR-Cas9 method, based on gene knock-in and knock-out, is another that may improve the therapeutic effects of the cells via upregulation of the genes crucial for Tregs [170,171].…”