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Chagas disease,caused by the parasite Trypanosoma cruzi,affects millions of people globally. Unfortunately,the available treatment options,especially for the chronic stage of the disease,are suboptimal. Given the chronic nature of the disease and the elusive nature of the parasite,there is a high need for new and safer drugs that deliver sterile cure. Posaconazole was a promising lead in the drug discovery pipeline but ultimately failed in clinical trials due to patient relapses. This failure illustrates the need for a drug screening assay that can predict sterile cure by assessing recrudescence after treatment. Here,we used human induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) as host cells for T. cruzi. The iMACs were highly susceptible to infection by the parasites. By combining red fluorescent protein (RFP)-expressing iMACs with mNeonGreen-expressing T. cruzi,we were able to monitor the dynamics of the infection through live cell imaging. The activity of the compounds benznidazole and posaconazole was consistent with the results of an established infection system using mouse primary macrophages. The post-mitotic nature of iMACs makes them suitable host cells for long-term assays needed to assess recrudescence of parasites. Moreover,their human origin,stable genetic background,and capacity for genetic modification make the iMACs excellent host cells for studying host-pathogen interaction. Author summaryThe parasite Trypanosoma cruzi,the causative agent of Chagas disease,is a global health concern affecting millions each year. Infection with T. cruzi can cause chronic disease,often remaining asymptomatic for decades before resulting in severe cardiac or gastro-intestinal pathologies. To date,only benznidazole and nifurtimox are used for treatment of the infection,but both drugs are suboptimal for curing the chronic stage. Posaconazole showed great promise in preclinical studies but failed to achieve sterile cure in clinical trials,causing patient relapses. These disappointing results underline the need for drug screening assays able to predict sterile cure by evaluating recrudescence post-treatment. We used human induced pluripotent stem cell derived macrophages as host cells for T. cruzi and testing of trypanocidal compounds. This model can be used for long-term in vitro screening assays to find new drug candidates against Chagas disease. The human origin of these cells combined with the possibility of upscaling their production make them great host cells for drug screening campaigns. View Publication

Background: Recurrent/metastatic head and neck squamous cell carcinoma ((R/M) HNSCC) represents one of the most aggressive and immunosuppressive cancers. Despite the introduction of immune checkpoint inhibitors (ICIs),only a limited number of patients obtain long-term benefits. In (R/M) HNSCC patients,the antitumor immune response is defective,conferring resistance and promoting tumor progression. Therefore,the identification of novel biomarkers for superior clinical outcomes and easily accessible in standard clinical settings is still an unmet clinical need. Methods: Blood liquid biopsies obtained from (R/M) HNSCC patients undergoing pembrolizumab therapy (monotherapy or in combination with chemotherapy) were analyzed by flow cytometry to evaluate the levels of circulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs),at baseline and during therapy. Correlations between these immunosuppressive immune cell subsets and clinical parameters (clinical response rate,progression-free survival (PFS),overall survival (OS) and performance status (PS)) were performed. Results: Univariate analysis showed that before therapy,higher circulating levels of both CD137⁺Tregs and LOX-1⁺PMN-MDSCs,identified patients with significantly worse survival. Furthermore,CD137⁺Tregs resulted also positively correlated with worse PS,while high levels of LOX-1⁺PMN-MDSCs negatively affected response to pembrolizumab,with a significant increase in non-responsive patients during therapy. Interestingly,both CD137⁺Tregs as well as LOX-1⁺PMN-MDSCs exerted a higher immunosuppression on T cell proliferation than CD137−Tregs and LOX-1⁻PMN-MDSCs,respectively. Multivariate analysis revealed that the circulating LOX-1⁺PMN-MDSC subset resulted as an independent prognostic factor for survival by multivariate analysis,as confirmed in an independent validation cohort. Conclusions: The levels of blood circulating LOX-1⁺PMN-MDSCs may be proposed as non-invasive biomarkers to predict clinical outcomes of (R/M) HNSCC patients developing resistance to immunotherapy,improving patient selection and suggesting novel personalized therapies. View Publication

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However,such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here,we present SNIPE,a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells,which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations. Genome editing often requires marker genes for selection of edited cells. Here,the authors present SNIPE,a marker-free method that selects cells based on DNA sequence,enabling precise enrichment of edited cells and applications from evolutionary research to the elimination of cancer cells. View Publication

Induced pluripotent stem cells (iPSCs) are commonly used to model human genetic diseases. Two main strategies are used. The first involves making iPSC lines from individual cases with a disease,and the second involves making disease-relevant gene edits in established iPSC lines. Because generating gene-edited lines is time consuming and expensive,most studies begin with one starting iPSC stock line and evaluate several gene-edited sublines. The current studies focus on gene-editing to model Lesch–Nyhan disease (LND),which is caused by mutations in the HPRT1 gene. The same pathogenic c.508C>T edit was made in four well-established stock lines,and three gene-edited lines were isolated from each. RNA sequencing (RNAseq) was,then,used to evaluate the impact of the gene edit. Gene-edited lines were compared to their corresponding stock lines,as well as to each other. An aggregate analysis of all lines combined was also conducted to determine the most robust findings across all lines. Results from gene editing were further compared with iPSC lines derived from individual cases with LND,to determine how closely findings from gene editing match results obtained with case-derived lines. There were two main findings. First,the same gene edit has a different impact on gene expression when starting with different starting stock lines. Second,the gene editing strategy does not produce the same results as the case-derived strategy. Potential explanations for these differences are addressed,along with the relevance of these two different strategies for disease modeling. View Publication

The T-cell receptor (TCR) initiates T-lymphocyte activation,but the mechanism of TCR activation remains uncertain. Here,we present cryogenic electron microscopy structures for the unliganded and human leukocyte antigen (HLA)-bound human TCR–CD3 complex in nanodiscs that provide a native-like lipid environment. Distinct from the open and extended conformation seen in detergent,the unliganded TCR–CD3 in nanodiscs adopts two related closed and compacted conformations that represent its physiologic resting state in vivo. By contrast,the HLA-bound complex adopts the open and extended conformation,and conformation-locking disulfide mutants show that ectodomain opening is necessary for maximal ligand-dependent T-cell activation. These structures also reveal conformation-dependent protein–lipid and glycan–glycan interactions within the TCR. Together,these results establish allosteric conformational change during TCR activation,reveal avenues for immunotherapeutic engineering,and highlight the importance of native-like lipid environments for membrane protein structure determination. The T-cell receptor (TCR) activation mechanism has remained uncertain. Here,the authors present molecular structures for the apo and ligand-bound human TCR–CD3 complex in lipid nanodiscs,revealing large conformational changes during activation. View Publication

Human induced pluripotent stem cells (hiPSCs) offer significant potential for differentiation and research applications in cardiovascular diseases. When induced differentiated hiPSC-derived cardiomyocytes (hiPSC-CMs) are transplanted into the infarcted myocardial region,they exhibit extremely low survival rates and unsatisfactory therapeutic effects due to ischemia,hypoxia,and immune inflammation in the surrounding environment. To address this issue,we used Panax notoginseng saponin R1 (NGR1),which has demonstrated significant protective effects in prior research,to pretreat hiPSC-CMs before transplantation. Utilizing an in vitro H2O2 oxidative stress model and a nude mouse myocardial infarction (MI) model,we investigated the mechanism through which NGR1 pretreatment enhances the therapeutic efficacy of hiPSC-CM transplantation. The results revealed that the hiPSC-CMs expressed cTnT. NGR1 did not promote the proliferation of hiPSC-CMs but instead induced elevated levels of p-Akt protein in these cells. Compared to hiPSC-CM transplantation alone,transplantation of hiPSC-CMs pretreated with NGR1 exhibited higher ejection fraction (EF) and fractional shortening (FS) values,along with reduced infarct size and collagen deposition. Additionally,there were more HNA-positive cardiomyocytes in the cardiac tissue,fewer TUNEL-positive signals,and increased VWF-positive and Lyve1-positive signals. Furthermore,the gene expression levels of VEGFC,IGF-1,and SDF-1 were higher. Therefore,NGR1 pretreatment improves the survival of transplanted hiPSC-CMs in tissues,reduces myocardial apoptosis,enhances cardiac function,decreases infarct size and collagen deposition,promotes angiogenesis and lymphangiogenesis,and stimulates paracrine secretion. View Publication