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Wu, X.

Publications and source records attributed to Wu, X..

At least 433 records · Page 24Linked to original sources

Tracking and perceiving diverse motion signals: Directional biases in human smooth pursuit and perception

Human smooth pursuit eye movements and motion perception behave similarly when observers track and judge the motion of simple objects, such as dots. But moving objects in our natural environment are complex and contain internal motion. We ask how pursuit and perception integrate the motion of objects with motion that is internal to the object. Observers (n = 20) tracked a moving random-dot kinematogram with their eyes and reported the objects perceived direction. Objects moved horizontally with vertical shifts of 0, {+/-}3, {+/-}6, or {+/-}9{degrees} and contained internal dots that were static or moved {+/-}90{degrees} up/down. Results show that whereas pursuit direction was consistently biased in the direction of the internal dot motion, perceptual biases differed between observers. Interestingly, the perceptual bias was related to the magnitude of the pursuit bias (r = 0.75): perceptual and pursuit biases were directionally aligned in observers that showed a large pursuit bias, but went in opposite directions in observers with a smaller pursuit bias. Dissociations between perception and pursuit might reflect different functional demands of the two systems. Pursuit integrates all available motion signals in order to maximize the ability to monitor and collect information from the whole scene. Perception needs to recognize and classify visual information, thus segregating the target from its context. Ambiguity in whether internal motion is part of the scene or contributes to object motion might have resulted in individual differences in perception. The perception-pursuit correlation suggests shared early-stage motion processing or perception-pursuit interactions.

neuroscience↗

Functional evolution of SARS-COV-2 Spike protein: adaptation on translation and infection via surface charge of spike protein

The SARS-COV-2 virus, which causes the COVID-19, is rapidly accumulating mutations to adapt to the hosts. We collected SARS-COV-2 sequence data from the end of 2019 to April 2022 to analyze for their evolutionary features during the pandemic. We found that most of the SARS-COV-2 genes are undergoing negative purifying selection, while the spike protein gene (S-gene) is undergoing rapid positive selection. From the original strain to the alpha, delta and omicron variant types, the Ka/Ks of the S-gene increases, while the Ka/Ks within one variant type decreases over time. During the evolution, the codon usage did not evolve towards optimal translation and protein expression. In contrast, only S-gene mutations showed a remarkable trend on accumulating more positive charges. This facilitates the infection via binding human ACE2 for cell entry and binding furin for cleavage. Such a functional evolution emphasizes the survival strategy of SARS-COV-2, and indicated new druggable target to contain the viral infection. The nearly fully positively-charged interaction surfaces indicated that the infectivity of SARS-COV-2 virus may approach a limit.

genetics↗

Stromal Hippo-YAP signaling in stem cell niche controls intestinal homeostasis

Intestinal homeostasis is tightly regulated by the reciprocal interaction between gut epithelium and adjacent mesenchyme. The mammalian Hippo-YAP pathway is intimately associated with intestinal epithelial homeostasis and re-generation; however, its role in postnatal gut mesenchyme remains poorly defined. We find that, although removal of the core Hippo kinases Lats1/2 or activation of YAP in adult intestinal smooth muscle has largely no effect; Hippo-YAP signaling in Gli1/PDGFR-expressing intestinal stromal cells is critical to maintain the stem cell niche. We show that YAP/TAZ activation drives over-proliferation and suppresses smooth muscle actin expression in the niche-forming Gli1+ mesenchymal progenitors. In addition, mesenchymal YAP/TAZ activation disrupts the epithelial-mesenchymal crosstalk by promoting Wnt ligand production, leading to epithelial Wnt pathway activation. Our data also reveal that YAP/TAZ are upregulated in the stroma during DSS-induced injury and stromal YAP activation promotes intestinal epithelial regeneration. Altogether, our data identify an essential requirement for stromal Hippo-YAP signaling in the stem cell niche during intestinal homeostasis. HIGHTLIGHTSO_LILats1/2 control proliferation and differentiation of adult gut mesenchymal progenitors. C_LIO_LIMesenchymal YAP/TAZ promotes Wnt ligand production in intestinal stem cell niche. C_LIO_LIYAP/TAZ is up-regulated in the mesenchyme during intestinal injury. C_LIO_LIStromal YAP activation promotes intestinal epithelial regeneration. C_LI

developmental biology↗

Evaluating Stored Platelet Shape Change Using Imaging Flow Cytometry

Platelets are routinely stored at room temperature for 5-7 days before transfusion. Stored platelet quality is traditionally assessed by Kunickis morphology score. This method requires extensive training, experience, and is highly subjective. Moreover, the number of laboratories familiar with this technique is decreasing. Cold storage of platelets has recently regained interest because of potential advantages such as reduced bacterial growth and preserved function. However, platelets exposed to cold temperatures change uniformly from a discoid to a spherical shape, reducing the morphology score outcomes to a binary "spheres versus discs" during cooling. We developed a simpler, unbiased screening tool to measure temperature-induced platelet shape change using imaging flow cytometry. When reduced to two dimensions, spheres appear circular, while discs are detected on a spectrum from fusiform to circular. We defined circular events as having a transverse axis of > 0.8 of the longitudinal axis and fusiform events [≤] 0.8 of the longitudinal axis. We show that most EGTA-treated platelets and spherical beads are detected in the gate for circular events, while fresh platelets are found mainly in the gate for fusiform events. Using this assay, mouse and human platelets show a temperature and time-dependent, two-dimensional shape change from fusiform to circular, consistent with their three-dimensional change from discs to spheres. The method we describe here is a valuable tool for detecting shape change differences in response to agonists or temperature and will help screen for therapeutic measures to mitigate the cold-induced storage lesion. Plain language summaryO_ST_ABSWhat is the context?C_ST_ABSO_LIPlatelets for transfusion are currently stored for 5-7 days at room temperature, increasing the risk for bacterial growth C_LIO_LICold storage reduces the risk for bacterial growth but reduces circulation time C_LIO_LIStored platelet quality can be assessed by the light microscopy-based Morphology Score, first described in the 1970s C_LIO_LIDownsides of the Morphology Score include subjectivity, extensive training, and reduced availability in platelet laboratories. C_LI What is new?O_LIIn this study, we provide data showing that the Morphology score is reduced to a binary spheres versus discs response in cold-exposed platelets C_LIO_LIWe developed an imaging flow cytometry-based approach to quantify platelets response to cold based on the two-dimensional projection of the three-dimensional shapes, i.e., fusiform (discoid) versus circular (discoid and spherical) C_LIO_LIWe provide validation of this approach in mouse and human platelets C_LIO_LIWe provide evidence for increased temperature sensitivity in mouse platelets C_LI What is the impact?O_LIThis study provides an easy and unbiased tool for laboratories working on circumventing the cold-induced storage lesion or documenting spherical shape change in general C_LI

pathology↗

Soil nitrogen impacts the rhizosphere microbial community owing to microbial hitchhiking

Microbial hitchhiking demonstrates that some nonmotile microbes utilize trans-species motility to traverse their environment; however, whether driving forces, such as plants and nitrogen, affect microbial hitchhiking is not clear. In our study, we explored the effects of plants and nitrogen fertilizer on Bacillus-hitchhiking by setting filter membranes and different nitrogen fertilizer concentration gradients. In the experimental treatment, we added a filter membrane to the soil to prevent hitchhiking. In the absence of plants, nitrogen alone had little influence on motile bacteria and hitchhiking. However, Bacillus contents were significantly impacted by the nitrogen concentration when the plants were rooted, leading to a great variation in cell motility function according to the functional analysis in the soil microbial community. After applying the filter membrane, there were no significant differences in Bacillus contents, microbial community structure or cell motility functional abundance, which illustrated that hitchhiking impacted the microbial community. Our analysis of co-occurrence between bulk soil motile bacteria (Bacillus) and rhizosphere bacteria also confirmed this. The correlation between bulk soil motile bacteria and the rhizosphere microbial community was strong in the groups with suitable nitrogen concentrations without filter membranes and was weak at all nitrogen levels in the no-membrane treatments. Thus, we concluded that plants and different nitrogen doses synergistically altered the soil microbiome by hitchhiking, whose effect depends on nitrogen.

microbiology↗

DNA methylation Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic β-cell Heterogeneity

The molecular and functional heterogeneity of pancreatic {beta}-cells is well recognized. Pancreatic islets harbor a small subset of {beta}-cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of this sub-population within islets is essential for appropriate insulin secretion. However, the distinguishing characteristics of this subpopulation and the mechanisms that restrict TH expression in {beta}-cells are not known. Here, we define the specific molecular and metabolic characteristics of the TH+ {beta}-cells and show that TH expression in {beta}-cells is restricted by DNA methylation patterning during {beta}-cell lineage specification. Ablation of de novo DNA methyltransferase Dnmt3a in the pancreatic- and endocrine-progenitor lineages results in a dramatic increase in the proportion of TH+ {beta}-cells, while {beta}-cell specific ablation of Dnmt3a has no effect on this sub-population. We demonstrate that maintenance of Th promoter DNA methylation patterns is essential for its continued restriction in postnatal {beta}-cells, and that loss of DNA methylation dysregulates TH expression in {beta}-cells in response to chronic overnutrition, contributing to impairment of {beta}-cell identity. These data highlight the essential requirement of DNA methylation patterning in regulating endocrine cell fates, and reveal a novel role of DNA methylation in {beta}-cell heterogeneity.

developmental biology↗

Subtyping evaluation of Salmonella Enteritidis using SNP and core genome MLST with nanopore reads

Whole genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing such as Oxford Nanopore Technologies (ONT) presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. However, evaluation, standardization and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of ONT for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths under seven hours of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms.

microbiology↗

Deeply conserved super-enhancers maintain stem cell pluripotency in placental mammals

Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share a deep evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of the transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) which displayed deep conservation in placental mammals and are sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR/Cas9 approach severely impaired the proliferative potential and the ability to form undifferentiated colonies. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals. Significance statementSuper-enhancers (SEs) hold stronger power than regular enhancers to direct gene expression in the regulation of stem cell pluripotency. To dissect how pluripotency-associated SEs have evolved in mammals, we performed a systematic comparison of SEs among pigs, humans, and mice. Our analysis allowed the identification of three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that are highly conserved in Placentalia (accounting for 94% of mammals) as well as many species-specific SEs. All three SEs were sufficient to direct pluripotency-dependent gene expression and disruption of each conserved SE caused the loss of stem cell pluripotency. Our work highlights a small number of highly conserved SEs essential for the maintenance of pluripotency.

cell biology↗

A bacteriophage-based, highly efficacious, needle and adjuvant-free, mucosal COVID-19 vaccine

The authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected and effective in preventing COVID-19, but do not induce efficient mucosal immunity, or prevent viral transmission. We developed a bacteriophage T4-based, multicomponent, needle and adjuvant-free, mucosal vaccine by engineering spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of T4-COVID vaccine induced higher virus neutralization antibody titers against multiple variants, balanced Th1/Th2 antibody and cytokine responses, stronger CD4+ and CD8+ T cell immunity, and higher secretory IgA titers in sera and bronchoalveolar lavage with no effect on the gut microbiota, compared to vaccination of mice intramuscularly. The vaccine is stable at ambient temperature, induces apparent sterilizing immunity, and provides complete protection against original SARS-CoV-2 strain and its Delta variant with minimal lung histopathology. This mucosal vaccine is an excellent candidate for boosting immunity of immunized and/or as a second-generation vaccine for the unimmunized population.

microbiology↗

CircEML1 facilitates the steroid synthesis in follicular granulosa cells of chicken through sponging gga-miR-449a to release IGF2BP3 expression

Non-coding RNAs (ncRNAs) induced competing endogenous RNAs (ceRNA) play crucial roles in various biological process by regulating target gene expression. However, the studies of ceRNA networks in the regulation of ovarian ovulation process of chicken remains deficient compared to that in mammals. In the present study, it was revealed that circEML1 was differential expressed in hens ovarian tissue at different ages (15W, 20W, 30W and 68W) and identified as a loop structure from EML1 pre-mRNA, which promoted the expressions of CYP19A1 and StAR and the production of E2 and P4 in follicular granulosa cells (GCs) using qRT-PCR and ELISA. Furthermore, circEML1 was proved to serve as a sponge of gga-miR-449a to participate in the steroidogenesis using the dual luciferase reporter, RNA FISH assays, qRT-PCR and ELISA assays. In addition, we evaluated several potential target genes of gga-miR-449a and found that IGF2BP3 was targeted by gga-miR-449a and promoted steroidogenesis and E2/P4 secretions in GCs, which may act the regulatory role via mTOR/p38MAPK pathways. Meanwhile, we implemented a rescue experiment and demonstrated that gga-miR-449a reversed the promoting role of circEML1 on IGF2BP3 expression and steroidogenesis. Eventually, this study suggested that circEML1/gga-miR-449a/IGF2BP3 axis exerted an important role in the regulation of steroidogenesis and steroid hormones production possibly through mTOR/p38MAPK pathways in follicular GCs of chicken and may contribute a better understanding of ceRNA network in the modulatory mechanism of hens ovarian development and ovulation cycle.

molecular biology↗

Ribo-macs derived from nucleoli:big ribosome clusters in the cytoplasm of naive stem cells

Primed stem cells and naive stem cells are important for understanding early development, but their ribosomes have not been focused on. In this study, we find that big ribosome clusters named Ribo-macs exist in the cytoplasm of naive stem cells. Then, we prove that Ribo-macs are dynamic and physiological in the cytoplasm, and can synthesize proteins associated with biogenesis of endoplasmic reticulum and mitochondria. We also discover and demonstrate that Ribo-macs are the nucleoli, which of significance is promoting us to rethink our understanding of nucleoli. Besides, we reveal that Ribo-macs have a compatible relation with P-bodies and stress granules. In a word, all the results about Ribo-macs provide us with a new insight to understand how cells adapt quickly to environment.

developmental biology↗

Different astrocyte-to-neuron conversion tracing reporters affect results in mouse striatum under Ptbp1 knockdown

Conversion of astrocytes to neurons (AtN) is a promising potential strategy for the treatment of neurodegenerative diseases. Recent studies have reported that shRNA-, CasRx-, or ASO-mediated Ptbp1 suppression could reprogram resident astrocytes to neurons1-3. However, some groups have disputed the data interpretation of the reported AtN conversion events4-7. These controversies surrounding AtN conversion may due to differences in the astrocyte fate-mapping systems they applied from that in the original study, i.e., recombinant mouse strains with astrocyte specific reporter constructs versus AAV-based labeling systems. Here, we applied AAV-based tracing systems to label astrocytes with GFAP-driven HA-tagged Cas13X (AAV-GFAP::Cas13X-NLS-HA-sgPtbp1). Compared to GFAP-driven tdTomato reporter (AAV-GFAP::tdTomato) system in previous studies, we found no AtN conversion in mouse striatum. Furthermore, no intermediate neurons and no neuron density increase suggested no AtN conversion. Our findings indicated that inconsistent AtN outcomes may arise from different fate-mapping systems between AAV and transgenic mice, as well as through use of different reporter proteins. Thus, the complexity of astrocyte labeling systems warrants careful attention when drawing conclusions about whether AtN conversion occurs.

neuroscience↗

Novel Murine models of Mania and Depression

Neuropathological mechanisms triggering manic syndrome or manic episodes in bipolar disorder remain poorly characterised, as the research progress is severely limited by the paucity of appropriate animal models. Here we developed a novel manic mice model by combining a series of chronic unpredictable rhythm disturbances (CURD), which include disruption of circadian rhythm, sleep deprivation, exposure to cone light, with subsequent interference of followed spotlight, stroboscopic illumination, high temperature stress, noise disturbance and foot shock. To validate this novel manic model, we used multiple behavioural and cell biology approaches comparing the CURD-model with healthy controls and depressed mice. The depression model was created by an exposure to an improved chronic unpredictable mild stress, which we defined as chronic unpredictable mild restraint (CUMR). A novel manic mice model induced by environmental stressors and free from genetic or pharmacological interventions will benefit research into pathological mechanisms of mania.

animal behavior and cognition↗

No evidence of induced skin cancer or other skin abnormalities after long term (66 week) chronic exposure to 222-nm far-UVC radiatio

Far-UVC radiation, typically defined as 200-235 nm, has similar or greater anti-microbial efficacy compared to conventional 254-nm germicidal radiation. In addition, biophysical considerations of the interaction of far-UVC with tissue, as well as multiple short-term safety studies in animal models and humans, suggest that far-UVC exposure may be safe for skin and eye tissue. Nevertheless, the potential for skin cancer after chronic long-term exposure to far-UVC has not been studied. Here, we assessed far-UVC induced carcinogenic skin changes and other pathological dermal abnormalities in 96 SKH-1 hairless mice of both sexes that were exposed to average daily dorsal skin doses of 396 mJ/cm2, 126 mJ/cm2 or 56 mJ/cm2 of 222 nm far-UVC radiation for 66 weeks, 5 days per week, 8 hours per day, as well as similarly-treated unexposed controls. No evidence for increased skin cancer, abnormal skin growths, or incidental skin pathology findings was observed in the far-UVC exposed mice. In addition, there were no significant changes in morbidity or mortality. The findings from this study support the long-term safety of long-term chronic exposure to far-UVC radiation, and therefore its potential suitability as a practical anti-microbial approach to reduce airborne viral and bacterial loads in occupied indoor settings.

cancer biology↗

Tracking SARS-CoV-2 Omicron diverse spike gene mutations identifies multiple inter-variant recombination events

The current pandemic of COVID-19 is fueled by more infectious emergent Omicron variants. Ongoing concerns of emergent variants include possible recombinants, as genome recombination is an important evolutionary mechanism for the emergence and re-emergence of human viral pathogens. Although recombination events among SARS-CoV-1 and MERS-CoV were well-documented, it has been difficult to detect the recombination signatures in SARS-CoV-2 variants due to their high degree of sequence similarity. In this study, we identified diverse recombination events between two Omicron major subvariants (BA.1 and BA.2) and other variants of concern (VOCs) and variants of interest (VOIs), suggesting that co-infection and subsequent genome recombination play important roles in the ongoing evolution of SARS-CoV-2. Through scanning high-quality completed Omicron spike gene sequences, eighteen core mutations of BA.1 variants (frequency >99%) were identified (eight in NTD, five near the S1/S2 cleavage site, and five in S2). BA.2 variants share three additional amino acid deletions with the Alpha variants. BA.1 subvariants share nine common amino acid mutations (three more than BA.2) in the spike protein with most VOCs, suggesting a possible recombination origin of Omicron from these VOCs. There are three more Alpha-related mutations (del69-70, del144) in BA.1 than BA.2, and therefore BA.1 may be phylogenetically closer to the Alpha variant. Revertant mutations are found in some dominant mutations (frequency >95%) in the BA.1 subvariant. Most notably, multiple additional amino acid mutations in the Delta spike protein were also identified in the recently emerged Omicron isolates, which implied possible recombination events occurred between the Omicron and Delta variants during the on-going pandemic. Monitoring the evolving SARS-CoV-2 genomes especially for recombination is critically important for recognition of abrupt changes to viral attributes including its epitopes which may call for vaccine modifications.

microbiology↗

Distinct denitrifying phenotypes of predominant bacteria mould nitrous oxide metabolism in upland agricultural soils

Denitrifying nitrous oxide (N2O) emissions in agroecosystems result from variations in microbial composition and soil properties. However, the microbial mechanisms of differential N2O emissions in agricultural soils are less understood. Microcosm experiments of two types of Chinese farmland soil were conducted with nitrate (250 mg/kg) and a combination of glucose (1000 mg/kg) and nitrate, and a case with no addition was used as the control. The results show that N2O accumulation in black soil (BF) was significantly higher than that in fluvo-aquic soil (FF) independent of carbon and nitrogen supply. The abundance of denitrifying genes was significantly higher in FF, but the ratios of genes responsible for N2O production (narG, nirS, and nirK) to the gene for N2O reduction (nosZ) did not significantly differ between the two soils. However, the soils showed obvious discrepancies in denitrifying bacterial communities. High accumulation of N2O was verified by the isolates of Rhodanobacter, which is predominant in BF due to its truncated denitrifying genes and lack of N2O reduction capacity. The dominance of complete denitrifiers such as Castellaniella in FF led to a rapid reduction in N2O and reduced N2O accumulation, as demonstrated when its corresponding isolate was inoculated into both studied soils. Therefore, the different phenotypes of N2O metabolism of the distinct denitrifiers maintained in the two soils caused their differing N2O accumulation. This knowledge could guide the regulation of the denitrifying bacterial community and the phenotypes of N2O metabolism in agricultural soils to reduce N2O emissions.

microbiology↗

Alternative 3' UTRs contributes to post-transcriptional gene expression regulation under high salt stress

High salt stress continually challenges growth and survival of many plants, but the underlying molecular basis is not fully explored. Alternative polyadenylation (APA) produces mRNAs with different 3'UTRs (alternative 3'UTRs) to regulate gene expression at post-transcriptional level. However, the roles of such process in response to salt stress remain elusive. Here, we reported that alternative 3'UTRs responded to high salt stress in the halophyte-Spartina alterniflora, which tolerant to hash salt environment. High salt stress induced the global APA and increased the prevalence of APA events. Strikingly, high salt stress significantly led to 3' UTR lengthening of 207 transcripts through increasing the usage of distal poly(A) sites. Transcripts with alternative 3' UTRs were mainly enriched in salt stress related ion transporters. Alternative 3' UTRs of SaHKT1 increased RNA stability and protein synthesis in vivo. Regulatory AU-rich elements were identified in the alternative 3' UTRs and alternative 3' UTRs increased protein level of SaHKT1 in an AU-rich element dependent manner. Finally, 3' UTR lengthening might result from variations in poly(A) signals and poly(A) factors. Overall, these results suggest that APA is a potential novel high salt stress responsive mechanism by modulating mRNA 3' UTR length. These results also reveal complex regulator roles of alternative 3' UTRs coupling alternative polyadenylation and regulatory elements at post-transcriptional level in plants. One sentence summaryAlternative 3' UTRs acts as a potential novel mechanism in gene expression regulation of high salt tolerant genes

plant biology↗

Balancing activation and costimulation of CAR tunes signaling dynamics and enhances therapeutic potency

BackgroundPrimary human T cells engineered with chimeric antigen receptors (CARs) ex vivo can be adoptively transferred to treat cancer. CD19-targeting CAR with CD28 costimulatory domain and CD3{zeta} activation domain have been approved by the US FDA for treating B cell malignancies. MethodsHere we generated mutation of immunorecpetor tyrosine-based activation motifs (ITAMs) in CD3{zeta}, namely 1XX CAR, which altered the balance of activation and costimulation. Next we investigated whether 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their WT counterparts. ResultsEven though 1XX CARs decreased cytotoxicity against tumor cells in vitro, they showed better anti-tumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells proliferated more in response to antigen stimulation in vitro, persisted longer in vivo and had higher percentage of central memory cells. As 1XX modification directly calibrates CAR activation potential, we utilized fluorescence resonance energy transfer (FRET)-based biosensor to monitor signaling dynamics downstream of CARs. Decreased ITAM numbers in 1XX resulted in similar ZAP70 activation, while 1XX induced higher Ca2+ elevation and faster Erk activation than WT CAR, which may contribute to the better therapeutic potency of 1XX. ConclusionsOur results established the surpiosity of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical application of 1XX CARs against solid tumors.

immunology↗