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RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However,protein and CRISPR RNA (crRNA) delivery in human cells can be challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation. View Publication

Understanding human hematopoietic stem cell fate control is important for its improved therapeutic manipulation. Asymmetric cell division,the asymmetric inheritance of factors during division instructing future daughter cell fates,was recently described in mouse blood stem cells. In human blood stem cells,the possible existence of asymmetric cell division remained unclear because of technical challenges in its direct observation. Here,we use long-term quantitative single-cell imaging to show that lysosomes and active mitochondria are asymmetrically inherited in human blood stem cells and that their inheritance is a coordinated,nonrandom process. Furthermore,multiple additional organelles,including autophagosomes,mitophagosomes,autolysosomes,and recycling endosomes,show preferential asymmetric cosegregation with lysosomes. Importantly,asymmetric lysosomal inheritance predicts future asymmetric daughter cell-cycle length,differentiation,and stem cell marker expression,whereas asymmetric inheritance of active mitochondria correlates with daughter metabolic activity. Hence,human hematopoietic stem cell fates are regulated by asymmetric cell division,with both mechanistic evolutionary conservation and differences to the mouse system. View Publication

AIMS Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies,systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers,and as such qualify the method as local inflammation model for clinical pharmacology studies. METHODS Eighteen healthy male volunteers received 2 or 4 intradermal 5 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion,skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate. RESULTS LPS elicited a visible response and returned to baseline at 48??hours. Erythema,perfusion and temperature were statistically significant (P  View Publication

Despite the growing knowledge of T cell responses in COVID-19 patients,there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here,with a predicted peptide library from SARS-CoV-2 S and N proteins,we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein,N361-369 (KTFPPTEPK),was the most dominant epitope from our selected peptide library. Importantly,we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants,as to the wild-type epitope. Further,the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells,to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2,and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally,this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains. View Publication

Acute coronary syndrome (ACS) is the most severe clinical manifestation of coronary heart disease.We performed an epigenome-wide analysis of circulating CD4+ and CD8+ T cells isolated from ACS patients and healthy subjects (HS),enrolled in the DIANA clinical trial,by reduced-representation bisulphite sequencing (RRBS). In CD4+ T cells,we identified 61 differentially methylated regions (DMRs) associated with 57 annotated genes (53% hyper- and 47% hypo-methylated) by comparing ACS patients vs HS. In CD8+ T cells,we identified 613 DMRs associated with 569 annotated genes (28% hyper- and 72% hypo-methylated) in ACS patients as compared to HS. In CD4+vs CD8+ T cells of ACS patients we identified 175 statistically significant DMRs associated with 157 annotated genes (41% hyper- and 59% hypo-methylated). From pathway analyses,we selected six differentially methylated hub genes (NFATC1,TCF7,PDGFA,PRKCB,PRKCZ,ABCA1) and assessed their expression levels by q-RT-PCR. We found an up-regulation of selected genes in ACS patients vs HS (P < 0.001). ABCA1,TCF7,PDGFA,and PRKCZ gene expression was positively associated with CK-MB serum concentrations (r = 0.75,P = 0.03; r = 0.760,P = 0.029; r = 0.72,P = 0.044; r = 0.74,P = 0.035,respectively).This pilot study is the first single-base resolution map of DNA methylome by RRBS in CD4+ and CD8+ T cells and provides specific methylation signatures to clarify the role of aberrant methylation in ACS pathogenesis,thus supporting future research for novel epigenetic-sensitive biomarkers in the prevention and early diagnosis of this pathology. View Publication

Staphylococcal enterotoxins (SE) pose a great threat to human health due to their ability to bypass antigen presentation and activate large amounts of conventional T cells resulting in a cytokine storm potentially leading to toxic shock syndrome. Unconventional T- and NK cells are also activated by SE but the mechanisms remain poorly understood. In this study,the authors aimed to explore the underlying mechanism behind SE-mediated activation of MAIT-,?? T-,and NK cells in vitro. CBMC or PBMC were stimulated with the toxins SEA,SEH,and TSST-1,and cytokine and cytotoxic responses were analyzed with ELISA and flow cytometry. All toxins induced a broad range of cytokines,perforin and granzyme B,although SEH was not as potent as SEA and TSST-1. SE-induced IFN-$\gamma$ expression in MAIT-,?? T-,and NK cells was clearly reduced by neutralization of IL-12,while cytotoxic compounds were not affected at all. Kinetic assays showed that unconventional T cell and NK cell-responses are secondary to the response in conventional T cells. Furthermore,co-cultures of isolated cell populations revealed that the ability of SEA to activate ?? T- and NK cells was fully dependent on the presence of both monocytes and $\alpha$$\beta$ T cells. Lastly,it was found that SE provoked a reduced and delayed cytokine response in infants,particularly within the unconventional T and NK cell populations. This study provides novel insights regarding the activation of unconventional T- and NK cells by SE,which contribute to understanding the vulnerability of young children towards Staphylococcus aureus infections. View Publication

BACKGROUND Circular RNA of vimentin (circ-VIM) is a predictor for poor prognosis of acute myeloid leukemia,but we had little information on its function in esophageal cancer (EC). Here we examined the effects of circ-VIM together with sevoflurane on immune escape and multiple oncogenic activities of EC. METHODS Bioinformatic tools,luciferase assay,and RNA immunoprecipitation were used to examine regulations between circ-VIM,miR-124-3p (miR-124),and PD-L1. CCK-8,wound healing,and Transwell assays were used to measure cell proliferation,migration,and invasion,respectively. The impacts of EC cells on cytotoxicity,proliferation,and apoptosis of CD8+ T cells were examined using LDH assay,CFSE staining,and Annexin V/PI staining,respectively. The in vivo tumorigenesis and lung metastases were assessed using xenograft model and tail vein injection of EC cells. RESULTS Significant upregulation of circ-VIM and PD-L1 and downregulation of miR-124 were detected in EC tissues or cells. Circ-VIM sponged miR-124 and released its suppression on the downstream target PD-L1. Sevoflurane,independent of circ-VIM,also upregulated miR-124 to lower PD-L1 expression. By modulating miR-124/PD-L1 axis,silencing circ-VIM and applying sevoflurane both inhibited immune escape and multiple oncogenic activities of EC in vitro,and suppressed xenograft growth and lung metastases in vivo. The inactivation of Ras/ERK signaling pathway was involved in suppression of malignant phenotypes by silencing circ-VIM and sevoflurane treatment. CONCLUSIONS Silencing circ-VIM and applying sevoflurane,by separately regulating miR-124/PD-L1 axis,presented synergistic effects in inhibiting immune escape and multiple malignant phenotypes of EC cells. View Publication

Systemic sclerosis (SSc) is a life-threatening chronic connective tissue disease with the characteristics of skin fibrosis,vascular injury,and inflammatory infiltrations. Though inhibition of phosphodiesterase 4 (PDE4) has been turned out to be an effective strategy in suppressing inflammation through promoting the accumulation of intracellular cyclic adenosine monophosphate (cAMP),little is known about the functional modes of inhibiting PDE4 by apremilast on the process of SSc. The present research aimed to investigate the therapeutic effects and underlying mechanism of apremilast on SSc. Herein,we found that apremilast could markedly ameliorate the pathological manifestations of SSc,including skin dermal thickness,deposition of collagens,and increased expression of $\alpha$-SMA. Further study demonstrated that apremilast suppressed the recruitment and activation of macrophages and T cells,along with the secretion of inflammatory cytokines,which accounted for the effects of apremilast on modulating the pro-fibrotic processes. Interestingly,apremilast could dose-dependently inhibit the activation of M1 and T cells in vitro through promoting the phosphorylation of CREB. In summary,our research suggested that inhibiting PDE4 by apremilast might provide a novel therapeutic option for clinical treatment of SSc patients. View Publication

Clever-1 also known as Stabilin-1 and FEEL-1 is a scavenger molecule expressed on a subpopulation of anti-inflammatory macrophages and lymphatic endothelial cells (LECs). However,its role in regulating dendritic cell (DC) trafficking and subsequent effects on immunity have remained unexplored. In this study,we demonstrate that DC trafficking from the skin into the draining lymph nodes is compromised in the absence of Clever-1. By adoptive transfer approaches we further show that the poor trafficking is due to the impaired entrance of DCs into afferent lymphatics. Despite this,injections of ovalbumin-loaded DCs into the footpads induced a stronger proliferative response of OT II T cells in the draining lymph nodes. This could be explained by the increased MHC II expression on DCs and a less tolerogenic phenotype of LECs in lymph nodes of Clever-1 knockout mice. Thus,although fewer DCs reach the nodes,they are more active in creating antigen-specific immune responses. This suggests that the DCs migrating to the draining lymph node within Clever-1 positive lymphatics experience immunosuppressive interactions with LECs. In conclusion,besides being a trafficking molecule on lymphatic vasculature Clever-1 is immunosuppressive towards migrating DCs and thus,regulates the magnitude of immune responses created by incoming DCs in the draining lymph nodes. View Publication