Functional CRISPR dissection of gene networks controlling human regulatory T cell identity

Abstract

Human regulatory T (T reg ) cells are essential for immune homeostasis. The transcription factor (TF) FOXP3 maintains T reg cell identity, yet the complete set of key TFs that control T reg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein (RNP) screens to identify TFs that regulate critical proteins in primary human T reg cells under basal and pro-inflammatory conditions. We then generated 54,424 single-cell transcriptomes from T reg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network co-regulated by FOXO1 and IRF4. We also discovered that HIVEP2, not previously implicated in T reg cell function, co-regulates another gene network with SATB1 and is important for T reg cell-mediated immunosuppression. By integrating CRISPR screens and scRNA-seq profiling, we have uncovered novel transcriptional regulators and downstream gene networks in human T reg cells that could be targeted for immunotherapies.

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

“…Another group used lentiviral siRNA to achieve FOXP3 knockdown in human Tregs, finding reduced in vitro suppressive function (Amendola et al, 2009). More recently, CRISPR-based FOXP3 KO in human Tregs revealed increased expression of IFN-γ, IL-4, and IL-2 and a trend towards reduced in vivo suppressive function (Schumann et al, 2020). In our system, FOXP3 HDR-KO Tregs did not significantly upregulate IL-2.…”

“…In contrast, FOXP3 ablation predominantly decreased DNA methylation: >95% of FOXP3 KO-induced DMRs were less methylated relative to Cas9 nTregs ( Figure 7C, Figure S5A). A number of genes associated with these FOXP3 KOinduced demethylated DMRs, including BACH2, HIVEP2, DUSP4, and RPTOR (mTORC1), have been implicated in mouse Treg development and/or function ( Figure 7C) (Roychoudhuri et al, 2013;Zeng et al, 2013;Yan et al, 2015;Schumann et al, 2020). We thus used a publicly available FOXP3 ChIP-chip tiled array dataset from human Tregs (Sadlon et al, 2010) to ask if any DMRs overlapped a FOXP3-binding region ( Table S1).…”

“…The use of CRISPR and CRISPR-mediated HDR in primary human Tregs has so far been limited. CRISPR/Cas9 editing in human Tregs with either RNP or lentivirus yielded a range of editing efficiencies (30-80%) dependent on target and gRNA (Chen et al, 2018;Noel et al, 2018;Seki and Rutz, 2018;Schumann et al, 2020). Combining CRISPR with a HDR ssDNA template, one group reported partial restoration of suppressive capacity in Tregs by correcting an IL2RA mutation (Roth et al, 2018).…”

Transcriptional Control of Regulatory T Cells in Cancer: Toward Therapeutic Targeting?

“…In addition, our knowledge will undoubtedly be improved by the implementation of large-scale screening technologies. For instance, three recent studies have used genome-wide and pooled Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screen approaches, to identify unknown elements involved in the maintenance of Foxp3 expression and Treg cell identity both in murine and human cells [128][129][130]. These reports have highlighted a series of novel pathways, such as ubiquitin ligases and deubiquitinases, chromatin remodelers, and transcription factors; in particular, Loo et al nicely demonstrated that ablation of the Bromodomain-containing protein 9 (Brd9) subunit of the non-canonical BRG1/BRM Associated Factors (BAF) complex, impaired Foxp3 expression and reduced Treg cell function, leading to enhanced tumor immunity and delayed growth of transplanted MC38 colon adenocarcinoma cells [129].…”

Critical cancer vulnerabilities identified by unbiased CRISPR/Cas9 screens inform on efficient cancer Immunotherapy

“…And while cancer immunotherapies have not been the primary focus for other CRISPR screens, this technology has been utilized to interrogate regulatory processes in B cells [105][106][107], macrophages [108][109][110][111][112][113], and dendritic cells [114][115][116]. Furthermore, the power this technology holds has unveiled novel gene networks controlling the function of specific subsets of T cells (e.g., Treg cells) that could be harnessed to increase anti-tumor immunity [117,118]. These studies highlight the potential that CRISPR-Cas9 technology holds to understand tumor biology, with the ultimate aim of improving the efficacy of current immunotherapeutic approaches or unveiling novel checkpoints in other immune cell populations.…”