Biology subjects

Liu, W.

Publications and source records attributed to Liu, W..

At least 325 records · Page 18Linked to original sources

bioRxiv2022

Quantitative profiling of pseudouridylation landscape in the human transcriptome

Pseudouridine ({Psi}) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, transcriptome-wide measurement of individual {Psi} sites remains unaddressed. Here, we develop "PRAISE", via selective chemical labeling of {Psi} by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the {Psi} landscape in the human transcriptome. Unlike traditional RNA/DNA bisulfite treatment, our approach is based on quaternary base mapping and revealed a ~10% median modification level for 2,714 confident {Psi} sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7 and TRUB1, with TRUB1 mRNA targets showing the highest modification stoichiometry. In addition, we identified and quantified known and novel {Psi} sites in mitochondrial mRNA, catalyzed by a mitochondria-localized isoform of PUS1. Collectively, we provide a sensitive and convenient method to measure transcriptome-wide {Psi}; we envision this quantitative approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.

molecular biology↗

bioRxiv2022

Microsecond pulse electrical stimulation modulates cell migration

Wound healing is a complicated process for maintaining skin integrity after injury, for which electrical stimulations (ES) are ascribed to promote wound healing by facilitating cell migration. Time-shortening of the stimulation treatment from current hours to minutes for efficient wound healing but free of cell damage in return, is however rather a challenge. Here, a novel mechanism of ultrashort pulse electric field (PEF), microsecond PEF at higher voltage, is proposed and realized to promote wound healing under a much short time (seconds) for the total treatment. We revealed that microsecond PEF regulated actin cytoskeleton reorganization and focal adhesion turnover, promoting fibroblasts migration in 2D cell cultures under the pulse stimulation. This accelerated fibroblast migration was accompanied by the mutual promotion with extracellular matrix (ECM) alignment in 3D microenvironments, which cooperatively benefit the eventual wound healing, and these findings were further confirmed by the enhanced skin wound healing in a classic mouse model. Additionally, we coined an actin- and collagen-dependent mechanism of microsecond PEF-mediated wound healing. The quantitative mechanism proposed here for our novel microsecond pulse electric filed (sPEF) methodology orients the new practical electric treatment in a wide range of biomedical applications, such as wound healing, regenerative medicine, and tissue engineering.

bioengineering↗

bioRxiv2022

A germline-targeting chimpanzee SIV envelope glycoprotein elicits a new class of V2-apex directed cross-neutralizing antibodies

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germline-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, non-neutralizing antibodies revealed that SCIV-expressed Envs as well as some primary HIV-1 Envs adopted a more open conformation, thereby exposing a conserved V2 epitope that is occluded in closed SIVcpz and HIV-1 Env trimers. These results expand the spectrum of V2-apex targeted antibodies that can contribute to neutralization breadth and identify novel SIV Env platforms for further development as germline-targeting and immunofocusing immunogens. One sentence summaryA cryptic V2 epitope in occluded-open HIV and SIV Env trimers is the target of a new class of V2-directed cross-neutralizing antibodies.

immunology↗

bioRxiv2022

Abundant Aβ fibrils in ultracentrifugal supernatants of aqueous extracts from Alzheimer's disease brains

Soluble aggregates of amyloid-{beta} (A{beta}), often called oligomers, are believed to be principal drivers of neurotoxicity, spreading of pathology, and symptoms in Alzheimers disease (AD), but little is known about their structures in human brain. A{beta} oligomers have been defined as aggregates found in supernatants following ultracentrifugation of aqueous extracts. We now report the unexpected presence of abundant A{beta} fibrils in high-speed supernatants from AD brains that were extracted by soaking in aqueous buffer. The fibrils did not appear to form during extract preparation, and their numbers by EM correlated with ELISA quantification of aggregated A{beta}42. Cryo-EM structures of A{beta} fibrils from aqueous extracts were identical to those from sarkosyl-insoluble AD brain homogenates. The fibrils in aqueous extracts were immunolabeled by lecanemab, an A{beta} aggregate-directed antibody reported to improve cognitive outcomes in AD. We conclude that A{beta} fibrils are abundant in aqueous extracts from AD brains and have the same structures as those from amyloid plaques. These findings have implications for understanding the nature of A{beta} oligomers and for designing oligomer-preferring therapeutic antibodies.

neuroscience↗

bioRxiv2022

RabbitTClust: enabling fast clustering analysis of millions bacteria genomes with MinHash sketches

We present RabbitTClust, a fast and memory-efficient genome clustering tool based on sketch-based distance estimation. Our approach enables efficient processing of large-scale datasets by combining dimensionality reduction techniques with streaming and parallelization on modern multi-core platforms. 113,674 complete bacterial genome sequences (RefSeq: 455 GB in FASTA format) can be clustered within less than 6 minutes and 1,009,738 GenBank assembled bacterial genomes (4.0 TB in FASTA format) within only 34 minutes on a 128-core workstation. Our results further identify 1,269 repetitive genomes (identical nucleotide content) in RefSeq bacterial genomes.

bioinformatics↗

bioRxiv2022

Simultaneous widefield voltage and interferometric dye-free optical mapping quantifies electromechanical waves in human iPSC-cardiomyocytes.

Coupled electro-mechanical waves define hearts function in health and disease. Genetic abnormalities, drug-triggered or acquired pathologies can disrupt and uncouple these waves with potentially lethal consequences. Optical mapping of electrical waves using fluorescent dyes or genetically-encoded sensors in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) offers mechanistic insights into cardiac conduction abnormalities. Interferometric dye-free/label-free wave mapping (without specific sensors) presents an alternative, likely capturing the mechanical aspects of cardiac conduction. Because of its non-invasive nature and spectral flexibility (not restricted to a specific excitation wavelength), it is an attractive chronic imaging tool in iPSC-CMs, as part of all-optical high-throughput platforms. In this study, we developed simultaneous widefield voltage and interferometric dye-free optical imaging methodology that was used: 1) to validate dye-free optical mapping for quantification of cardiac wave properties in human iPSC-CMs; 2) to demonstrate low-cost optical mapping of electromechanical waves in hiPSC-CMs using recent near-infrared (NIR) voltage sensors and orders of magnitude cheaper miniature CMOS cameras; 3) to uncover previously underexplored frequency- and space-varying parameters of cardiac electromechanical waves in hiPSC-CMs. We find similarity in the frequency-dependent responses of electrical (NIR fluorescence imaged) and mechanical (dye-free imaged) waves, with the latter being more sensitive to faster rates and showing steeper restitution and earlier appearance of wave-front tortuosity. During regular pacing, the dye-free imaged conduction velocity and the electrical wave velocity are correlated; both modalities being sensitive to pharmacological uncoupling and both dependent on gap-junctional protein (connexins) determinants of wave propagation. We uncover strong frequency-dependence of the electromechanical delay (EMD) locally and globally in hiPSC-CMs on a rigid substrate. The presented framework and results offer new means to track the functional responses of hiPSC-CM inexpensively and non-invasively for counteracting heart disease and aiding cardiotoxicity testing and drug development.

bioengineering↗

bioRxiv2022

Functional genomics of cattle through integration of multi-omics data

Functional annotation of the bovine genome was performed by characterizing the spectrum of RNA transcription using a multi-omics approach, combining long- and short-read transcript sequencing and orthogonal data to identify promoters and enhancers and to determine boundaries of open chromatin. A total number of 171,985 unique transcripts (50% protein-coding) representing 35,150 unique genes (64% protein-coding) were identified across tissues. Among them, 159,033 transcripts (92% of the total) were structurally validated by independent datasets such as PacBio Iso-seq, ONT-seq, de novo assembled transcripts from RNA-seq, or Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive independent data from different technologies such as WTTS-seq, RAMPAGE, ChIP-seq, and ATAC-seq. A large proportion of identified transcripts (69%) were novel, of which 87% were produced by known genes and 13% by novel genes. A median of two 5 untranslated regions was detected per gene, an increase from Ensembl and NCBI annotations (single). Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as non-coding genes in fetal tissues, but as protein coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 known gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available QTL data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. These validated results show significant improvement over current bovine genome annotations.

bioinformatics↗

bioRxiv2022

Irx1 and Irx2 play dose-dependent cooperative functions in mammalian development

Irx1 and Irx2 (Irx1/2) are two closely linked and widely expressed members of the conserved Iroquois homeobox family of transcription factors. Despite mounting evidence suggesting the importance of homologs of these genes in many aspects of vertebrate development and function, the role of Irx1/2 in mammals has remained largely unknown. Here, we used mice carrying our newly generated Irx1flox and Irx1floxIrx2del mutant alleles to perform a stepwise genetic ablation of Irx1 and Irx2 levels. Our analysis revealed reduced postnatal growth and viability of Irx1KO mice with gross histological defects in the lung and gut and demonstrated that ablation of one copy of Irx2 in these mice results in neonatal lethality with exacerbated phenotypic defects. Conversely, while Irx2KO mice appear normal, ablation of one copy of Irx1 in these mutants leads to lethality at weaning. Furthermore, we found that homozygous deletion of both Irx1 and Irx2 results in embryonic lethality by mid-gestation with defective extraembryonic vasculature. Our results illustrate that Irx1 and Irx2 play distinct dose-dependent cooperative functions during both the early and late stages of mouse development.

developmental biology↗

bioRxiv2022

Intranasal delivery of NS1-deleted influenza virus vectored COVID-19 vaccine restrains the SARS-CoV-2 inflammatory response

The emergence of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) variants and "anatomical escape" characteristics threaten the effectiveness of current coronavirus disease (COVID-19) vaccines. There is an urgent need to understand the immunological mechanism of broad-spectrum respiratory tract protection to guide broader vaccines development. In this study, we investigated immune responses induced by an NS1-deleted influenza virus vectored intranasal COVID-19 vaccine (dNS1-RBD) which provides broad-spectrum protection against SARS-CoV-2 variants. Intranasal delivery of dNS1-RBD induced innate immunity, trained immunity and tissue-resident memory T cells covering the upper and lower respiratory tract. It restrained the inflammatory response by suppressing early phase viral load post SARS-CoV-2 challenge and attenuating pro-inflammatory cytokine (IL-6, IL-1B, and IFN-{gamma}) levels, thereby reducing excess immune-induced tissue injury compared with the control group. By inducing local cellular immunity and trained immunity, intranasal delivery of NS1-deleted influenza virus vectored vaccine represents a broad-spectrum COVID-19 vaccine strategy to reduce disease burden.

immunology↗

bioRxiv2022

Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the [~]190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs. HighlightsO_LIDe novo design, construction and functional characterization of a neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae. C_LIO_LIIncreasing the copy number of the 275 highly expressed tRNA genes causes cellular burden, which the host cell likely buffers either by selecting for partial tRNA neochromosome deletions or by increasing its ploidy. C_LIO_LIThe tRNA neochromosome can be chemically extracted and transformed into new strain backgrounds, enabling its transplantation into multi-synthetic chromosome strains to finalize the Sc2.0 strain. C_LIO_LIComprehensive functional characterization does not pinpoint a singular cause for the cellular burden caused by the tRNA neochromosome, but does reveal novel insights into its tRNA and structural chromosome biology. C_LI

synthetic biology↗

bioRxiv2022

DEED: A Dataset for Dream-related Emotion Research

Emotion is closely related to human cognition and behaviour. In recent years, scholars have conducted extensive research on emotion in waking state based on electroencephalography (EEG) and achieved certain results. However, Emotional activity continues after sleep, with a concentrated response of sleep emotions in dreams. Sleep emotions are concentrated in dreams, which can better reflect a series of real physical and psychological states of the human body. Currently, there is no publicly available dataset for the assessment of dream mood. Therefore, we present a physiological dataset Dream Emotion Evaluation Dataset (DEED) for the assessment of dream mood, which recorded EEG signals from 38 participants over 89 whole sleep nights and 533 dream segments(after exclusion of unqualified nights, those dream segments are extracted from 82 whole sleep nights). We studied the correlations between the subjective ratings and the EEG signals and brain network patterns for dream emotions. In addition, the relationship between the asymmetry of left and right brain bands and positive and negative dream emotions was studied. The machine learning algorithm was also used to classify different emotional EEG, which confirmed the validity of the dataset. In the meantime, we encourage other researchers to explore the underlying neural mechanisms involved in sleep.

neuroscience↗

bioRxiv2022

SRSF2 is required for mRNA splicing and spermatogenesis

RNA splicing plays significant roles in fundamental biological activities. However, our knowledge about the roles of alternative splicing and underlying mechanisms during spermatogenesis is limited. Here, we report that Serine/arginine-rich splicing factor 2 (SRSF2), also known as SC35, plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf2 by Stra8-Cre caused complete infertility and defective spermatogenesis. Further analyses revealed that deletion of Srsf2 disrupted differentiation and meiosis initiation of spermatogonia. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF2 regulatory networks play critical roles in several major events including reproductive development, spermatogenesis, meiotic cell cycle, synapse organization, DNA recombination, chromosome segregation, and male sex differentiation. Furthermore, SRSF2 affected expression and alternative splicing of Stra8, Stag3 and Atr encoding critical factors for spermatogenesis in a direct manner. Taken together, our results demonstrate that SRSF2 has important functions in spermatogenesis and male fertility by regulating alternative splicing.

developmental biology↗

bioRxiv2022

Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis

Histone chaperones and chromatin remodelers control nucleosome dynamics, which are essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display a preference for the HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefers the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated histone deposition for proper epigenetic regulation of the genome.

plant biology↗

bioRxiv2022

Identification of the Metaphyseal Skeletal Stem Cell

Identification of novel regional skeletal stem cells (SSCs) will provide a new cellular paradigm for bone physiology and dysfunction. Several populations of SSCs have been identified at distinct skeletal sites. However, a bona fide SSC population has not yet been formally characterized in the bone marrow. Here, we identify a metaphyseal SSCs (mpSSCs) population whose transcriptional landscape is distinct from other bone mesenchymal stromal cells (bMSCs) in the bone marrow. These mpSSCs emerge at the postnatal stage and reside just underneath the growth plate, consistent with the fact that these mpSSCs are exclusively derived from hypertrophic chondrocytes (HCs). These mpSSCs possess SSC properties such as self-renewal and multipotency in vitro and in vivo, stand at the top of the HC de-differentiation path, and produce most HC progeny. Genetic block of the conversion from HCs to mpSSCs significantly compromises trabecular bone formation and bone regeneration. Thus, metaphysis houses a unique HC-derived SSC population, which is a major source of osteoblasts and bMSCs supporting postnatal trabecular bone formation.

developmental biology↗

bioRxiv2022

The cytidine deaminase APOBEC3G drives cancer mutagenesis and clonal evolution in bladder cancer

Mutagenic processes leave distinct signatures in cancer genomes. The mutational signatures attributed to APOBEC3 cytidine deaminases are pervasive in human cancers. However, data linking individual APOBEC3 proteins to cancer mutagenesis in vivo are limited. Here, we show that transgenic expression of human APOBEC3G promotes mutagenesis, genomic instability, and kataegis, leading to shorter survival in a murine bladder cancer model. Acting as mutagenic fuel, APOBEC3G increases the clonal diversity of bladder cancers, driving divergent cancer evolution. We characterize the single base substitution signature induced by APOBEC3G in vivo, showing the induction of a mutational signature different from that caused by APOBEC3A and APOBEC3B. Analysis of thousands of human cancers reveals the contribution of APOBEC3G to the mutational profiles of multiple cancer types, including bladder cancer. Our findings define the role of APOBEC3G in cancer mutagenesis and clonal heterogeneity. These results potentially inform future therapeutic efforts that restrict tumor evolution.

genomics↗

bioRxiv2022

Construction of epithelial-mesenchymal transition related miRNAs signatures as prognostic biomarkers in gastric cancer patients

AimTo identify the potential post-healing EMT related miRNAs associated with lymph node metastatic gastric cancer (LNMGC). MethodsBoth RNA expression and clinical medical data were obtained from the TCGA dataset. We performed differential expression and normalization analysis of miRNAs. Cox linear regression model confirmed the differentially expressed miRNAs (DEmiRNAs) and clinical medical parameters related to overall survival (OS). The role of target genes of DEmiRNAs was determined according to the role enrichment analysis. ResultsWe obtained a total of 7531 DEmRNAs and 267 DEmRNAs, of which 185 DEmRNAs were down-regulated and 82 DEmRNAs were up-regulated. We randomly divided the LMNGC cases (n=291) into a training group (n=207) and a test group (n=84). The results showed that a total of 103, 11, 13 and 83 overlapping genes were associated with hsa-mir-141-3p, hsa-mir-4664-3p, hsa-mir-125b-5p and hsa-mir-7-5p, respectively. Kaplan-Meier determined that these four miRNAs can effectively distinguish high-risk and low-risk groups, and have a good indicator role (all p<0.05). Multivariate cox regression analysis also showed that EMT-related miRNA predictive model and lymph node metastasis were both prognostic risk factors (all p<0.05). The ROC curve showed that this feature had high accuracy (AUC>0.7, p<0.05). In addition, KEGG analysis showed that EMT-related pathways were mainly enriched in HIF-1 signaling pathway and focal adhesion. ConclusionsOur study demonstrated that EMT-related miRNAs could serve as independent prognostic markers in pN1-3 GC patients.

cancer biology↗

bioRxiv2022

Mapping mitonuclear epistasis using a novel recombinant yeast population

Natural genetic variation in mitochondrial and nuclear genomes can influence phenotypes by perturbing coadapted mitonuclear interactions. Mitonuclear epistasis, i.e. non-additive phenotype effects of interacting mitochondrial and nuclear alleles, is emerging as a general feature in eukaryotes, yet very few mitonuclear loci have been identified. Here, we present a novel advanced intercrossed population of S. cerevisiae yeasts, called the Mitonuclear Recombinant Collection (MNRC), designed explicitly for detecting mitonuclear loci contributing to complex traits, and use this population to map the genetic basis to mtDNA loss. In yeast, spontaneous deletions within mtDNAs lead to the petite phenotype that heralded mitochondrial research. We show that in natural populations, rates of petite formation are variable and influenced by genetic variation in nuclear, mtDNAs and mitonuclear interactions. We then mapped nuclear and mitonuclear alleles contributing mtDNA stability using the MNRC by integrating mitonuclear epistasis into a genome-wide association model. We found that associated mitonuclear loci play roles in mitotic growth most likely responding to retrograde signals from mitochondria, while associated nuclear loci with main effects are involved in genome replication. We observed a positive correlation between growth rates and petite frequencies, suggesting a fitness tradeoff between mitotic growth and mtDNA stability. We also found that mtDNA stability was influenced by a mobile mitochondrial GC-cluster that is expanding in certain populations of yeast and that selection for nuclear alleles that stabilize mtDNA may be rapidly occurring. The MNRC provides a powerful tool for identifying mitonuclear interacting loci that will help us to better understand genotype-phenotype relationships and coevolutionary trajectories. Author SummaryGenetic variation in mitochondrial and nuclear genomes can perturb mitonuclear interactions and lead to phenotypic differences between individuals and populations. Despite their importance to most complex traits, it has been difficult to identify the interacting loci. Here, we created a novel population of yeast designed explicitly for mapping mitonuclear loci contributing to complex traits and used this population to map genes influencing the stability of mitochondrial DNA (mtDNA). We found that mitonuclear interacting loci were involved in mitotic growth while non-interacting loci were involved in genome replication. We also found evidence that selection for mitonuclear loci that stabilize mtDNAs occurs rapidly. This work provides insight into mechanisms underlying maintenance of mtDNAs. The mapping population presented here is an important new resource that will help to understand genotype/phenotype relationships and coevolutionary trajectories.

evolutionary biology↗

bioRxiv2022

Spike-specific CXCR3+ TFH cells play a dominant functional role in supporting antibody responses in SARS-CoV-2 infection and vaccination

CD4+ T follicular helper (TFH) cells are required for high-quality antibody generation and maintenance. However, the longevity and functional role of these cells are poorly defined in COVID-19 convalescents and vaccine recipients. Here, we longitudinally investigated the dynamics and functional roles of spike-specific circulating TFH cells and their subsets in convalescents at the 2nd, 5th, 8th, 12th and 24th months after COVID-19 symptom onset and in vaccinees after two and three doses of inactivated vaccine. SARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, of which spike-specific CXCR3+ TFH cells but not spike-specific CXCR3- TFH cells and neutralizing antibodies were persistent for at least two years in more than 80% of convalescents who experienced symptomatic COVID-19, which was well coordinated between spike-specific TFH cell and antibody responses at the 5th month after infection. Inactivated vaccine immunization also induced spike-specific TFH cell and antibody responses; however, these responses rapidly declined after six months with a two-dose standard administration, and a third dose significantly promoted antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells exhibited better responsiveness than spike-specific CXCR3- TFH cells upon spike protein stimulation in vitro and showed superior capacity in supporting spike-specific antibody secreting cell (ASC) differentiation and antibody production than spike-specific CXCR3- TFH cells cocultured with autologous memory B cells. In conclusion, spike-specific CXCR3+ TFH cells played a dominant functional role in antibody elicitation and maintenance in SARS-CoV-2 infection and vaccination, suggesting that induction of CXCR3-biased spike-specific TFH cell differentiation will benefit SARS-CoV-2 vaccine development aiming to induce long-term protective immune memory. HighlightsO_LISARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, which persisted for at least two years in the majority of symptomatic COVID-19 convalescent patients. C_LIO_LIInactivated vaccine immunization also elicited spike-specific TFH cell and antibody responses, which rapidly declined over time, and a third dose significantly promoted antibody maturation and potency. C_LIO_LISpike-specific CXCR3+ TFH cells exhibited more durable responses than spike-specific CXCR3- TFH cells, correlated with antibody responses and showed superior capacity in supporting ASC differentiation and antibody production than spike-specific CXCR3- TFH cells. C_LI

immunology↗