The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3–10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Endoplasmic reticulum (ER) stress is a physiological response to protein overload or misfolded proteins in the ER. Certain anti-cancer drugs, e.g. bortezomib and nelfinavir, induce ER stress implying that this could be a successful therapeutic strategy against several forms of cancer. To find novel ER-stress inducers we screened a panel of natural and synthetic Toll-like receptor (TLR) agonists against human keratinocytes and identified the anti-cancer drug imiquimod (IMQ) as a potent inducer of ER stress. Other TLR7 and TLR8 agonists, including resiquimod and gardiquimod, did not induce ER stress, demonstrating that IMQ induces ER stress independently of TLR7 and TLR8. We further confirmed this by showing that IMQ could still induce ER stress in mouse Tlr7(-/-) cells. IMQ also induced a rapid and transient influx of extracellular Ca(2+) together with the release of Ca(2+) from internal stores. Depletion of Ca(2+) from the ER is a known cause of ER stress suggesting that IMQ induces ER stress via depletion of ER Ca(2+). The ER-stress inducing property of IMQ is possibly of importance for its efficacy in treating basal cell carcinoma, in situ melanoma, and squamous cell carcinoma. Our data could potentially be harnessed for rational design of even more potent ER-stress inducers and new anti-cancer drugs.

Chronic stimulation can cause T cell dysfunction and limit efficacy of cellular immunotherapies. CRISPR screens have nominated gene targets for engineered T cells, but improved methods are required to compare large numbers of synthetic knockin sequences to reprogram cell functions. Here, we developed Modular Pooled Knockin Screening (ModPoKI), an adaptable platform for modular construction of DNA knockin libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors. Over 20 ModPoKI screens across human TCR and CAR T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct to enhance long-term T cell fitness and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate a ~10,000-member library of TF combinations. Non-viral knockin of a combined BATF-TFAP4 polycistronic construct further enhanced function in vivo. ModPoKI facilitates discovery of complex gene constructs to program cellular functions.

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TRIM21 is an interferon-stimulated E3 ligase that controls the activity of pattern-recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone-marrow-derived macrophages (BMDMs) generated from Trim21 À/À mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony-stimulating factor (M-CSF), Trim21 À/À BMDMs had decreased expression of M-CSF signature genes. Although Trim21 À/À BMDMs responded normally to Toll-like receptor 9 (TLR9) activation, they produced lower levels of pro-inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette-Gu erin strain of Mycobacterium bovis was also diminished in Trim21 À/À BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists.

Systemic autoimmune diseases are characterized by the overexpression of type I IFN stimulated genes, and accumulating evidence indicate a role for type I IFNs in these diseases. However, the underlying mechanisms for this are still poorly understood. To explore the role of type I IFN regulated miRNAs in systemic autoimmune disease, we characterized cellular expression of miRNAs during both acute and chronic type I IFN responses. We identified a T cell-specific reduction of miR-31-5p levels, both after intramuscular injection of IFNβ and in patients with Sjögren's syndrome (SjS). To interrogate the role of miR-31-51p in T cells we transfected human CD4 + T cells with a miR-31-5p inhibitor and performed metabolic measurements. This identified an increase in basal levels of glucose metabolism after inhibition of miR-31-5p. Furthermore, treatment with IFN-α also increased the basal levels of human CD4 + T-cell metabolism. In all, our results suggest that reduced levels of miR-31-5p in T cells of SjS patients support autoimmune T-cell responses during chronic type I IFN exposure.Keywords: Autoimmunity r Interferons r Immune regulation r Metabolism r Rheumatology Additional supporting information may be found online in the Supporting Information section at the end of the article.by the overexpression of a large set of IFN-stimulated genes (ISGs) collectively termed the IFN signature [1,2]. Type I IFNs signal through the heterodimeric IFNA receptor and induce the expression of ISGs, several of which have important antiviral * These authors contributed equally.

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