Deletion mapping of regulatory elements for GATA3 in T cells reveals a distal enhancer involved in allergic diseases.

GATA3 is essential for T cell differentiation and is surrounded by genome-wide association study (GWAS) hits for immune traits. Interpretation of these GWAS hits is challenging because gene expression quantitative trait locus (eQTL) studies lack power to detect variants with small effects on gene expression in specific cell types and the genome region containing GATA3 contains dozens of potential regulatory sequences. To map regulatory sequences for GATA3, we performed a high-throughput tiling deletion screen of a 2 Mb genome region in Jurkat T cells. This revealed 23 candidate regulatory sequences, all but one of which is within the same topological-associating domain (TAD) as GATA3. We then performed a lower-throughput deletion screen to precisely map regulatory sequences in primary T helper 2 (Th2) cells. We tested 25 sequences with ∼100 bp deletions and validated five of the strongest hits with independent deletion experiments. Additionally, we fine-mapped GWAS hits for allergic diseases in a distal regulatory element, 1 Mb downstream of GATA3, and identified 14 candidate causal variants. Small deletions spanning the candidate variant rs725861 decreased GATA3 levels in Th2 cells, and luciferase reporter assays showed regulatory differences between its two alleles, suggesting a causal mechanism for this variant in allergic diseases. Our study demonstrates the power of integrating GWAS signals with deletion mapping and identifies critical regulatory sequences for GATA3.

CRISPR Screening of CAR T Cells and Cancer Stem Cells Reveals Critical Dependencies for Cell-Based Therapies.

Glioblastoma (GBM) contains self-renewing GBM stem cells (GSC) potentially amenable to immunologic targeting, but chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited clinical responses in GBM. Here, we interrogated molecular determinants of CAR-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. Screening of CAR T cells identified dependencies for effector functions, including TLE4 and IKZF2. Targeted knockout of these genes enhanced CAR antitumor efficacy. Bulk and single-cell RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered tumor-immune signaling and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs discovered avenues for enhancing CAR therapeutic efficacy against GBM, with the potential to be extended to other solid tumors. SIGNIFICANCE: Reciprocal CRISPR screening identified genes in both CAR T cells and tumor cells regulating the potency of CAR T-cell cytotoxicity, informing molecular targeting strategies to potentiate CAR T-cell antitumor efficacy and elucidate genetic modifications of tumor cells in combination with CAR T cells to advance immuno-oncotherapy.This article is highlighted in the In This Issue feature, p. 995.

Three-dimensional bioprinted glioblastoma microenvironments model cellular dependencies and immune interactions.

Brain tumors are dynamic complex ecosystems with multiple cell types. To model the brain tumor microenvironment in a reproducible and scalable system, we developed a rapid three-dimensional (3D) bioprinting method to construct clinically relevant biomimetic tissue models. In recurrent glioblastoma, macrophages/microglia prominently contribute to the tumor mass. To parse the function of macrophages in 3D, we compared the growth of glioblastoma stem cells (GSCs) alone or with astrocytes and neural precursor cells in a hyaluronic acid-rich hydrogel, with or without macrophage. Bioprinted constructs integrating macrophage recapitulate patient-derived transcriptional profiles predictive of patient survival, maintenance of stemness, invasion, and drug resistance. Whole-genome CRISPR screening with bioprinted complex systems identified unique molecular dependencies in GSCs, relative to sphere culture. Multicellular bioprinted models serve as a scalable and physiologic platform to interrogate drug sensitivity, cellular crosstalk, invasion, context-specific functional dependencies, as well as immunologic interactions in a species-matched neural environment.

TGF-ß-responsive CAR-T cells promote anti-tumor immune function.

A chimeric antigen receptor (CAR) that responds to transforming growth factor beta (TGF-ß) enables the engineering of T cells that convert this immunosuppressive cytokine into a potent T-cell stimulant. However, clinical translation of TGF-ß CAR-T cells for cancer therapy requires the ability to productively combine TGF-ß responsiveness with tumor-targeting specificity. Furthermore, the potential concern that contaminating, TGF-ß?producing regulatory T (Treg) cells may preferentially expand during TGF-ß CAR-T cell manufacturing and suppress effector T (Teff) cells demands careful evaluation. Here, we demonstrate that TGF-ß CAR-T cells significantly improve the anti-tumor efficacy of neighboring cytotoxic T cells. Furthermore, the introduction of TGF-ß CARs into mixed T-cell populations does not result in the preferential expansion of Treg cells, nor do TGF-ß CAR-Treg cells cause CAR-mediated suppression of Teff cells. These results support the utility of incorporating TGF-ß CARs in the development of adoptive T-cell therapy for cancer.

Rewiring T-cell responses to soluble factors with chimeric antigen receptors.

Chimeric antigen receptor (CAR)-expressing T cells targeting surface-bound tumor antigens have yielded promising clinical outcomes, with two CD19 CAR-T cell therapies recently receiving FDA approval for the treatment of B-cell malignancies. The adoption of CARs for the recognition of soluble ligands, a distinct class of biomarkers in physiology and disease, could considerably broaden the utility of CARs in disease treatment. In this study, we demonstrate that CAR-T cells can be engineered to respond robustly to diverse soluble ligands, including the CD19 ectodomain, GFP variants, and transforming growth factor beta (TGF-ß). We additionally show that CAR signaling in response to soluble ligands relies on ligand-mediated CAR dimerization and that CAR responsiveness to soluble ligands can be fine-tuned by adjusting the mechanical coupling between the CAR's ligand-binding and signaling domains. Our results support a role for mechanotransduction in CAR signaling and demonstrate an approach for systematically engineering immune-cell responses to soluble, extracellular ligands.