Biology subjects

Van Gool, F.

Publications and source records attributed to Van Gool, F..

5 recordsLinked to original sources

bioRxiv2020

The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge (HD) and transmembrane (TMD) domains between the extracellular antigen-targeting and the intracellular signaling modalities of CARs has not been systemically studied. Here, a series of CD19-CARs differing only by their HD (CD8/CD28/IgG4) and TMD (CD8/CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation to anti-CD28 stimulation. This dimerization depended on polar amino-acids in the CD28-TMD. CD28-CAR heterodimerization was more efficient in CARs containing a CD8-HD or CD28-HD as compared to an IgG4-HD. CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but led to a significant reduction of CD28 cell-surface expression. These data unveil a new property of the CD28-TMD and suggest that TMDs can modulate CAR T-cell activities by engaging endogenous partners. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/296913v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@bcd17aorg.highwire.dtl.DTLVardef@5cbca9org.highwire.dtl.DTLVardef@660eecorg.highwire.dtl.DTLVardef@5db163_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

CRISPR Screen in Regulatory T Cells Reveals Ubiquitination Modulators of Foxp3

Regulatory T cells (Tregs) are required to control immune responses and maintain homeostasis but are a significant barrier to anti-tumor immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of pro-inflammatory properties2, can promote autoimmunity and/or facilitate more effective tumor immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. Despite improved functional genetic tools that now allow for systematic interrogation, dissection of the gene regulatory programs that modulate Foxp3 expression has not yet been reported. In this study, we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Tregs and applied this technology to perform a targeted loss-of-function screen of [~]490 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We discovered several novel modulators including ubiquitin-specific peptidase 22 (Usp22), Ataxin 7 like 3 (Atxn7l3) and ring finger protein 20 (Rnf20). Members of the deubiquitination module of the SAGA chromatin modifying complex, Usp22 and Atxn7l3, were discovered to be positive regulators that stabilized Foxp3 expression; whereas the screen suggested Rnf20, an E3 ubiquitin ligase, is a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein and created defects in their suppressive function that led to spontaneous autoimmunity but protected against tumor growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Tregs could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Tregs. These results reveal novel modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

immunology↗

bioRxiv2019

Multiple environmental signaling pathways control the differentiation of RORγt-expressing regulatory T cells

ROR{gamma}t-expressing Tregs form a specialized subset of intestinal CD4+ Foxp3+ cells which is essential to maintain gut homeostasis and tolerance to commensal microbiota. Recently, c-Maf emerged as a critical factor in the regulation of ROR{gamma}t expression in Tregs. However, aside from c-Maf signaling, the signaling pathways involved in the differentiation of ROR{gamma}t+ Tregs and their possible interplay with c-Maf in this process are largely unknown. We show that ROR{gamma}t+ Treg development is controlled by positive as well as negative signals. Along with c-Maf signaling, signals derived from a complex microbiota, as well as IL-6/STAT3- and TGF-{beta}-derived signals act in favor of ROR{gamma}t+ Treg development. Ectopic expression of c-Maf did not rescue ROR{gamma}t expression in STAT3-deficient Tregs, indicating the presence of additional effectors downstream of STAT3. Moreover, we show that an inflammatory IFN-{gamma}/STAT1 signaling pathway acts as a negative regulator of ROR{gamma}t+ Treg differentiation in a c-Maf independent fashion.\n\nThese data thus argue for a complex integrative signaling network that finely tunes ROR{gamma}t expression in Tregs. The finding that type 1 inflammation impedes ROR{gamma}t+ Treg development even in the presence of an active IL-6/STAT3 pathway further suggests a dominant negative effect of STAT1 over STAT3 in this process.

immunology↗

bioRxiv2019

Thymically-derived Foxp3+ regulatory T cells are the primary regulators of type 1 diabetes in the non-obese diabetic mouse model

Regulatory T cells (Tregs) are an immunosuppressive population that are identified based on the stable expression of the fate-determining transcription factor forkhead box P3 (Foxp3). Tregs can be divided into distinct subsets based on whether they developed in the thymus (tTregs) or in the periphery (pTregs). Whether there are unique functional roles that distinguish pTregs and tTregs remains largely unclear. To elucidate these functions, efforts have been made to specifically identify and modify individual Treg subsets. Deletion of the conserved non-coding sequence (CNS)1 in the Foxp3 locus leads to selective impairment of pTreg generation without disrupting tTreg generation in the C57BL/6J background. Using CRISPR-Cas9 genome editing technology, we removed the Foxp3 CNS1 region in the non-obese diabetic (NOD) mouse model of spontaneous type 1 diabetes mellitus (T1D) to determine if pTregs contribute to autoimmune regulation. Deletion of CNS1 impaired in vitro induction of Foxp3 in naive NOD CD4+ T cells, but it did not alter Tregs in most lymphoid and non-lymphoid tissues analyzed except for the large intestine lamina propria, where a small but significant decrease in ROR{gamma}t+ Tregs and corresponding increase in Helios+ Tregs was observed in NOD CNS1-/- mice. CNS1 deletion also did not alter the development of T1D or glucose tolerance despite increased pancreatic insulitis in pre-diabetic female NOD CNS1-/- mice. CNS1 Furthermore, the proportions of autoreactive Tregs and conventional T cells (Tconvs) within pancreatic islets were unchanged. These results suggest that pTregs dependent on the Foxp3 CNS1 region are not the dominant regulatory population controlling T1D in the NOD mouse model.

immunology↗

bioRxiv2016

Discovery of an autoimmunity-associated IL2RA enhancer by unbiased targeting of transcriptional activation

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell type-specific transcriptional programs and responses to specific extracellular cues 1-3. In order to understand the mechanisms by which non-coding genetic variation contributes to disease, systematic mapping of functional enhancers and their biological contexts is required. Here, we develop an unbiased discovery platform that can identify enhancers for a target gene without prior knowledge of their native functional context. We used tiled CRISPR activation (CRISPRa) to synthetically recruit transcription factors to sites across large genomic regions (>100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA (interleukin-2 receptor alpha; CD25). We identified several CRISPRa responsive elements (CaREs) with stimulation-dependent enhancer activity, including an IL2RA enhancer that harbors an autoimmunity risk variant. Using engineered mouse models and genome editing of human primary T cells, we found that sequence perturbation of the disease-associated IL2RA enhancer does not block IL2RA expression, but rather delays the timing of gene activation in response to specific extracellular signals. This work develops an approach to rapidly identify functional enhancers within non-coding regions, decodes a key human autoimmunity association, and suggests a general mechanism by which genetic variation can cause immune dysfunction.

genetics↗