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Sequence-specific proteases have proven to be versatile building blocks for tools that report or control cellular function. Reporting methods link protease activity to biochemical signals, whereas control methods rely on engineering proteases to respond to exogenous inputs such as light or chemicals. In turn, proteases have inherent control abilities, as their native functions are to release, activate or destroy proteins by cleavage, with the irreversibility of proteolysis allowing sustained downstream effects. As a result, protease-based synthetic circuits have been created for diverse uses such as reporting cellular signaling, tuning protein expression, controlling viral replication and detecting cancer states. Here, we comprehensively review the development and application of protease-based methods for reporting and controlling cellular function in eukaryotes. This Review focuses on protease-based tools for sensing and controlling protein function in cell biology.

CD8+ T cells differentiate into diverse states that shape immune outcomes in cancer and chronic infection. To systematically define the transcription factors (TFs) driving these states, we built a comprehensive atlas integrating transcriptional and epigenetic data across nine CD8+ T cell states and inferred TF activity profiles. Our analysis catalogued TF activity fingerprints, uncovering regulatory mechanisms governing selective cell state differentiation. Leveraging this platform, we focused on two transcriptionally similar but functionally opposing states critical in tumor and viral contexts: terminally exhausted T cells (TEXterm), which are dysfunctional, and tissue-resident memory T cells (TRM), which are protective. Global TF community analysis revealed distinct biological pathways and TF-driven networks underlying protective versus dysfunctional states. Through in vivo CRISPR screening integrated with single-cell RNA sequencing (in vivo Perturb-seq), we delineated that TFs selectively govern TEXterm. We identified HIC1 and GFI1 as shared regulators of TEXterm and TRM differentiation and KLF6 as a unique regulator of TRM. Importantly, we discovered novel TEXterm single-state TFs, including ZSCAN20 and JDP2 with no prior known function in T cells. Targeted deletion of these TFs enhanced tumor control and synergized with immune checkpoint blockade. Consistently, their depletion in human T cells reduces the expression of inhibitory receptors and improves effector function. By decoupling exhaustion-selective from protective TRM programs, our platform enables more precise engineering of T cell states, advancing rational design of effective immunotherapies.

The same types of cells can assume diverse states with varying functionalities. Effective cell therapy can be achieved by specifically driving a desirable cell state, which requires the elucidation of key transcription factors (TFs). Here, we integrated epigenomic and transcriptomic data at the systems level to identify TFs that define different CD8+ T cell states in an unbiased manner. These TF profiles can be used for cell state programming that aims to maximize the therapeutic potential of T cells. For example, T cells can be programmed to avoid a terminal exhaustion state (TexTerm), a dysfunctional T cell state that is often found in tumors or chronic infections. However, TexTerm exhibits high similarity with the beneficial tissue-resident memory T states (TRM) in terms of their locations and transcription profiles. Our bioinformatic analysis predicted Zscan20, a novel TF, to be uniquely active in TexTerm. Consistently, Zscan20 knock-out thwarted the differentiation of TexTerm in vivo, but not that of TRM. Furthermore, perturbation of Zscan20 programs T cells into an effector-like state that confers superior tumor and virus control and synergizes with immune checkpoint therapy. We also identified Jdp2 and Nfil3 as powerful TexTerm drivers. In short, our multiomics-based approach discovered novel TFs that enhance anti-tumor immunity, and enable highly effective cell state programming.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Author list

Transcription factors (TFs) govern cell fate through coordinated gene-regulatory networks, yet the full potential of these networks to generate non-native, therapeutically advantageous cell states remains largely unexplored. We hypothesized that systematic gain-of-function (GOF) overexpression of TFs in CD8 T cells, central mediators of immune protection, could reveal latent, or \"hidden,\" regulatory programs capable of generating synthetic T cell states with therapeutic utility. To test this, we developed single-cell GOF sequencing (scGOF-seq), a multiplexed platform for unbiased, mapping of GOF effects on T cell fate in immunocompetent mouse models of infection and cancer. scGOF-seq uncovered unexpected regulators of T cell differentiation and accumulation, including SOX2, OCT4, and GATA2, which are normally silenced during T cell differentiation. Notably, outside its native regulatory context, supraphysiologic cMyc GOF reprogrammed CD8 T cells into a synthetic stem-effector hybrid state, enabling >5,000-fold antigen-dependent expansion and antitumor activity, contrasting sharply with its native function in driving terminal differentiation. scGOF-seq further identified TF modules that cooperate with cMyc GOF to promote robust CD8 T cell responses in solid tumors. Together, these findings establish GOF perturbation as a powerful strategy for revealing latent immune regulatory programs and engineering synthetic immune states with therapeutic potential.

Transcription factors (TFs) regulate the differentiation of T cells into diverse states with distinct functionalities. To precisely program desired T cell states in viral infections and cancers, we generated a comprehensive transcriptional and epigenetic atlas of nine CD8 + T cell differentiation states for TF activity prediction. Our analysis catalogued TF activity fingerprints of each state, uncovering new regulatory mechanisms that govern selective cell state differentiation. Leveraging this platform, we focused on two critical T cell states in tumor and virus control: terminally exhausted T cells (TEX term ), which are dysfunctional, and tissue-resident memory T cells (T RM ), which are protective. Despite their functional differences, these states share significant transcriptional and anatomical similarities, making it both challenging and essential to engineer T cells that avoid TEX term differentiation while preserving beneficial T RM characteristics. Through in vivo CRISPR screening combined with single-cell RNA sequencing (Perturb-seq), we validated the specific TFs driving the TEX term state and confirmed the accuracy of TF specificity predictions. Importantly, we discovered novel TEX term -specific TFs such as ZSCAN20, JDP2, and ZFP324. The deletion of these TEX term -specific TFs in T cells enhanced tumor control and synergized with immune checkpoint blockade. Additionally, this study identified multi-state TFs like HIC1 and GFI1, which are vital for both TEX term and T RM states. Furthermore, our global TF community analysis and Perturb-seq experiments revealed how TFs differentially regulate key processes in T RM and TEX term cells, uncovering new biological pathways like protein catabolism that are specifically linked to TEX term differentiation. In summary, our platform systematically identifies TF programs across diverse T cell states, facilitating the engineering of specific T cell states to improve tumor control and providing insights into the cellular mechanisms underlying their functional disparities.Transcription factors (TFs) regulate the differentiation of T cells into diverse states with distinct functionalities. To precisely program desired T cell states in viral infections and cancers, we generated a comprehensive transcriptional and epigenetic atlas of nine CD8 + T cell differentiation states for TF activity prediction. Our analysis catalogued TF activity fingerprints of each state, uncovering new regulatory mechanisms that govern selective cell state differentiation. Leveraging this platform, we focused on two critical T cell states in tumor and virus control: terminally exhausted T cells (TEX term ), which are dysfunctional, and tissue-resident memory T cells (T RM ), which are protective. Despite their functional differences, these states share significant transcriptional and anatomical similarities, making it both challenging and essential to engineer T cells that avoid TEX term differentiation while preserving beneficial T RM characteristics. Through in vivo CRISPR screening combined with single-cell RNA sequencing (Perturb-seq), we validated the specific TFs driving the TEX term state and confirmed the accuracy of TF specificity predictions. Importantly, we discovered novel TEX term -specific TFs such as ZSCAN20, JDP2, and ZFP324. The deletion of these TEX term -specific TFs in T cells enhanced tumor control and synergized with immune checkpoint blockade. Additionally, this study identified multi-state TFs like HIC1 and GFI1, which are vital for both TEX term and T RM states. Furthermore, our global TF community analysis and Perturb-seq experiments revealed how TFs differentially regulate key processes in T RM and TEX term cells, uncovering new biological pathways like protein catabolism that are specifically linked to TEX term differentiation. In summary, our platform systematically identifies TF programs across diverse T cell states, facilitating the engineering of specific T cell states to improve tumor control and providing insights into the cellular mechanisms underlying their functional disparities.

The limited efficacy of immunotherapies against glioblastoma illustrates the urgent need to better understand the interactions between the central nervous system and the immune system. Here, we showed that a protective response to αCTLA-4 therapy depended on a mutualistic relationship between microglia and CD4+ T cells. Suppression of gliomas by CD4+ T cells did not require tumor-intrinsic MHC-II expression, but rather was dependent on the selective expression of MHC-II and antigen presentation by local microglia that in turn, sustained CD4+ T cell tumoricidal effector functions. CD4+ T cell secretion of IFNγ made the glioma cells vulnerable to enhanced tumor surveillance and phagocytosis by microglia via the AXL/MER tyrosine kinase receptors that were necessary for tumor suppression. This work illustrates a novel partnership between CD4+ T cells and microglia that unleashes the tumoricidal properties of microglia that can be harnessed to improve immunotherapies for glioblastoma.

Evidence-based robotic intervention programmes for children with autism spectrum disorder (ASD) have been limited. As yet, there is insufficient evidence to inform therapists, teachers, and service providers on effectiveness of robotic intervention to enhance social development and participation of children with ASD in a real context. This study used a randomised controlled trial to test the efficacy of robotic intervention programmes in enhancing the social development and participation of children with ASD. 60 children with ASD were included. The participants were randomly assigned to the following groups: (1) robotic intervention programme ( n  = 20), (2) human-instructed programme ( n  = 20), and (3) control group ( n  = 20). Both the performance-based behavioural change in social communication and parent-reported change in social responsiveness were evaluated. The participants in the robotic intervention group demonstrated statistically significant changes in both the performance-based assessment and parent-reported change in social participation. Significant differences were found in the communication and reciprocal social interactions scores between the experimental group and the control and comparison groups in the performance-based assessment ( p  < 0.01). The effectiveness of robotic intervention programme to enhance the social communication and participation was confirmed. Future studies may also consider adding a maintenance phase to document how the effects of the intervention carry over to the participants over a longer period. (Clinical trial number: NCT04879303; Date of registration: 10 May 2021).

Excessive fructose intake presents the major risk factor for metabolic cardiovascular disease. Perivascular adipose tissue (PVAT) is a metabolic tissue and possesses a paracrine function in regulating aortic reactivity. However, whether and how PVAT alters vascular function under fructose overconsumption remains largely unknown. In this study, male Sprague-Dawley rats (8 weeks old) were fed a 60% high fructose diet (HFD) for 12 weeks. Fasting blood sugar, insulin, and triglycerides were significantly increased by HFD intake. Plasma adiponectin was significantly enhanced in the HFD group. The expression of uncoupling protein 1 (UCP1) and mitochondrial mass were reduced in the aortic PVAT of the HFD group. Concurrently, the expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and mitochondrial transcription factor A (TFAM) were suppressed. Furthermore, decreased fusion proteins (OPA1, MFN1, and MFN2) were accompanied by increased fission proteins (FIS1 and phospho-DRP1). Notably, the upregulated α-smooth muscle actin (α-SMA) and osteocalcin in the PVAT were concurrent with the impaired reactivity of aortic contraction and relaxation. Coenzyme Q 10 (Q, 10 mg/100 mL, 4 weeks) effectively reversed the aforementioned events induced by HFD. Together, these results suggested that the dysregulation of mitochondrial dynamics mediated HFD-triggered PVAT whitening to impair aortic reactivity. Fortunately, coenzyme Q 10 treatment reversed HFD-induced PVAT whitening and aortic reactivity.

Background Aortic valve stenosis (AS) is a common, lethal cardiovascular disease. There is no cure except the valve replacement at last stage. Therefore, an understanding of the detail mechanism is imperative to prevent and intervene AS. Metabolic syndrome (MetS) is one of the major risk factors of AS whereas fructose overconsuming tops the list of MetS risk factors. However, whether the fructose under physiological level induces AS is currently unknown. Methods The human valve interstitial cells (hVICs), a crucial source to develop calcification, were co-incubated with fructose at 2 or 20 mM to mimic the serum fructose at fasting or post-fructose consumption, respectively, for 24 h. The cell proliferation was evaluated by WST-1 assays. The expressions of osteogenic and fibrotic proteins, PI3K/AKT signaling, insulin receptor substrate 1 and mitochondrial dynamic proteins were detected by Western blot analyses. The mitochondrial oxidative phosphorylation (OXPHOS) was examined by Seahorse analyzer. Results hVICs proliferation was significantly suppressed by 20 mM fructose. The expressions of alkaline phosphatase (ALP) and osteocalcin were enhanced concurrent with the upregulated PI3K p85, AKT, phospho(p)S473-AKT, and pS636-insulin receptor substrate 1 (p-IRS-1) by high fructose. Moreover, ATP production capacity and maximal respiratory capacity were enhanced in the high fructose groups. Synchronically, the expressions of mitochondrial fission 1 and optic atrophy type 1 were increased. Conclusions These results suggested that high fructose stimulated the osteogenic differentiation of hVICs via the activation of PI3K/AKT/mitochondria signaling at the early stage. These results implied that high fructose at physiological level might have a direct, hazard effect on the progression of AS.

Background Elevation of systemic inflammatory markers were found to correlate with increased disease extent, reduced lung function and higher risk of future severe exacerbations in patients with bronchiectasis. Although a significant correlation of circulating hs-CRP levels with HRCT scores and resting oxygen saturation in patients with stable-state non-cystic fibrosis (CF) bronchiectasis was suggested, there is little data on the relationship between hs-CRP and the prognosis of bronchiectasis and a lack of data on the role of hs-CRP in predicting bronchiectasis exacerbation. Methods A prospective study was conducted on Chinese patients with non- CF bronchiectasis from 1st October to 31st December 2021. Baseline serum hs-CRP were obtained at stable-state. The follow-up period lasted for one year. Co-primary endpoints were the development of any bronchiectasis exacerbation and hospitalized bronchiectasis exacerbation. Results Totally 123 patients were included. Higher hs-CRP was associated with increased risk to develop any bronchiectasis exacerbation, adjusted odds ratio (aOR) of 2.254 (95% CI = 1.040–4.885, p  = 0.039), and borderline significantly increased hospitalized bronchiectasis exacerbation with aOR of 1.985 (95% CI = 0.922–4.277, p  = 0.080). Conclusion Baseline serum hs-CRP level at stable-state can predict risk of bronchiectasis exacerbation, which is reflecting chronic low-grade inflammation in bronchiectasis.