Abstract Background & Aims Chronic HBV infection usually causes cirrhosis and hepatocellular carcinoma. Comparative investigations of acute and chronic HBV cases would help determine the immune responses crucial for viral clearance. Methods A fast-cleared HBV mouse model was established in Alb-Cre mice via hydrodynamic injection of HBV plasmid, while persistent HBV model mice were generated via recombinant covalently closed circular DNA-adeno-associated virus 8 infection. The single-cell transcriptomes of CD45^+ intrahepatic non-parenchymal cells from these mice were conducted. Multiplexed immunohistochemistry and flow cytometry were used to confirm the findings from single-cell transcriptomes. Transwell, coculture, and adoptive transfer experiments were performed to study the generation and functions of macrophages. Results Twenty-four clusters of immune cells were identified. Myeloid cells, including granulocytes, monocytes, and dendritic cells, are activated early in HBV fast-cleared mice. Significantly, a cluster of CD3^+ macrophages was found in the viral clearance phase, which was confirmed in liver tissue from five acute patients with HBV. These cells highly expressed CXCL1, tumor necrosis factor alpha, and HBsAg-specific T cell receptors. The transwell assay revealed that CD3^+ macrophages originate from macrophages (n = 6). T cells and anti-HBsAg antibodies are indispensable for their differentiation, which was further confirmed in T- and/or B-cell-deficient mice. Interestingly, these CD3^+ macrophages capable of killing peptide-loaded hepatocytes and engulfing IgG-coated beads were persistently detectable in the mouse liver for 10 weeks after HBV clearance. The expression levels of CD5L and Bcl2, two classical antiapoptotic proteins, increased (p <0.001), suggesting that the CD3^+ macrophages are long-term resident populations. Finally, adoptive transfer of CD3^+ macrophages accelerated HBV clearance in mice (n = 5, p <0.01). Conclusions We identified long-term polyfunctional CD3^+ macrophages residing in HBV fast-cleared livers that could help elucidate the immune responses involved in eliminating HBV. Impact and implications The liver is a special organ with unique immune characteristics and tolerance to foodborne antigens. Chronic infections can develop in newborns after exposure to HBV; however, acute infections usually occur in adults, indicating that immune cells in the liver tissue microenvironment can also effectively fight against the virus. Nevertheless, the mechanisms involved in acute HBV infection have rarely been studied. In this study, we identified a macrophage population with both T cell and macrophage characteristics in the livers of acute HBV model mice and revealed that these macrophages play important roles in HBV clearance. Moreover, we confirmed that this population is derived from macrophages in the presence of virus-specific T cells and antibodies. This finding highlights the complexity of antiviral immune responses in liver microenvironments. Keywords: Liver, HBV clearance, CD3^+ macrophage, scRNA-seq Graphical abstract [43]Image 1 [44]Open in a new tab Highlights * • Exhausted CD8^+ T cell numbers increase in HBV-persistent livers, whereas myeloid cell numbers increase in fast-cleared livers. * • CD3^+ macrophages are a distinctive population that increases in the liver of HBV fast-cleared mice and acutely infected patients. * • CD3^+ macrophages originate from intrahepatic macrophages. * • CD3^+ macrophages are long-term liver-resident cells that promote HBV clearance. Introduction Hepatitis B, caused by hepatitis B virus (HBV) infection, is a major public health problem. More than 250 million people have chronic HBV infection and are at high risk of developing cirrhosis, liver failure, and hepatocellular carcinoma, which results in over 800,000 deaths per year.[45]^1 Viral infection in humans can lead to different clinical outcomes. Acute or self-limited infection, with the virus eventually eliminated, occurs in most adults exposed to HBV. In contrast, more than 90% of people exposed to HBV in early childhood develop chronic hepatitis B (CHB).[46]^2^,[47]^3 In a previous study, we revealed a potential mechanism for the establishment of chronic HBV infection via thymic-homed myeloid-derived suppressor cells[48]^4; however, it is not fully understood which immune cell populations are crucial for eliminating viruses in acute and self-limited hepatitis B. HBV is a hepatotropic virus,[49]^5 and the liver is also considered an immune organ with innate immune advantages because of its enrichment with immune cells such as Kupffer cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells (DCs), hepatic stellate cells (HSCs), sinusoidal endothelial cells (LSECs), mucosal-associated invariant T (MAIT) cells, B cells, and T cells.[50][6], [51][7], [52][8] Many studies have shown that impaired liver regional immunity is a reason for HBV chronicity, whereas effective immune responses lead to viral clearance in acute and self-limited patients with hepatitis B. In recent years, single-cell sequencing (scRNA-seq), an emerging technology,[53]^9^,[54]^10 has increased our ability to analyze the heterogeneity of immune cells in the tissue microenvironment. Identifying crucial cell populations helps reveal the mechanism of disease development; however, the heterogeneity, dynamic regulation, and tolerance mechanisms of various immune cells in the liver microenvironment have not been fully elucidated. Because an HBV infection model in immune-competent mice is still lacking, in this study, HBV fast-cleared model mice were established via hydrodynamic injection of the HBV pUC-B6 plasmid, as previously reported,[55]^11 and persistent HBV model mice were generated via the injection of adeno-associated virus 8 (AAV8) carrying recombinant covalently closed circular DNA (rcccDNA) into 6-week-old Alb-Cre mice, mimicking the situation of patients with intrahepatic HBV from CHB, as shown in our previous work.[56]^12 The transcription profiles of immune cells in the livers of HBV mice were subsequently analyzed via scRNA-seq technology. The heterogeneity of intrahepatic immune cells was investigated. By comparing the differences in the ecological landscapes of livers from persistent or fast-cleared HBV model mice, a new cluster of CD3^+ macrophages was identified as a population that can accelerate HBV clearance. Importantly, CD3^+ macrophages were also observed in tissues (biopsies) from the livers of patients with acute hepatitis B(AHB) but were not found in patients with CHB, indicating their important roles in viral clearance. Materials and methods Clinical specimens and mice The clinical liver biopsy procedure was performed using needles with a 1-mm inner diameter, and with the patients’ written consent. Liver tissues were then frozen embedded in optimal cutting temperature compound and cut into 8-μm sections. C57BL/6 and BALB/c mice were purchased from Super B&K Laboratory Animal Corp. Ltd. Rag1 knockout and nude mice were purchased from Jiangsu Jicui Pharmachem Bio-technology. MuMT knockout mice were kindly provided by Professor Jianhua Li from Fudan University. HBsAg T cell receptor (TCR) transgenic mice (Balb/c background) were purchased from The Jackson Lab (No. 027510). Alb-Cre transgenic mice (males, aged 8 weeks) expressing Cre recombinase under the albumin promoter were purchased at the Center of Laboratory Animals, Shanghai Public Health Clinical Center. Fast-cleared HBV mouse model was established via hydrodynamic injection as reported,[57]^11 whereas HBV-persistent mice were constructed by tail vein injection of rcccDNA-AAV8 virus in 6-week-old Alb-Cre mice.[58]^12 All mice were housed and bred under specific pathogen-free or BSL2 conditions. The Ethics Committee of Shanghai Medical College approved the use of liver tissue samples from human patients and the mouse experiments (No. 2022-C001). Quantification of HBsAg, HBeAg, and HBV DNA The mouse serum alanine aminotransferase (ALT), HBsAg, and HBeAg were quantified at Labway Clinical Laboratory (Shanghai, China) using Roche's Cobas e601. HBV DNA from mouse serum was determined by qPCR using a hepatitis B viral DNA Quantitative Fluorescence Diagnostic Kit according to the manufacturer's instructions (Sansure, Hunan, China). Preparation of single-cell suspensions Single-cell suspensions from five mouse livers in each group were prepared by enzyme digestion and tissue mincing. Briefly, mice were euthanized and subsequently perfused with HBSS containing 0.04% collagenase IV from the hepatic portal vein. Livers were isolated, minced, and filtered. The non-parenchymal cells were isolated using a Percoll gradient. CD45^+ cells were sorted using an ARIA II (BD, Biosciences). About 10,000 cells per sample with viability >80% were loaded. Single-cell RNA sequencing Single-cell RNA sequencing was conducted by Shanghai Bohao Biotechnology Co., Ltd. To capture single-cell transcriptomic information of CD45^+ immune cell samples, the BD Rhapsody system (BD Biosciences) was used as previously reported.[59]^13^,[60]^14 Single-cell capture was achieved by random distribution of a single-cell suspension across ∼200,000 microwells. Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation, so that each cell was paired with a bead in a microwell. After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA synthesis, each cDNA molecule was tagged on the 5′ end (that is, the 3′ end of a messenger RNA transcript) with UMI and cell label indicating its cell of origin. Whole-transcriptome libraries were prepared using the BD Resolve single-cell whole-transcriptome amplification workflow. Briefly, Rhapsody beads were then subject to second-strand cDNA synthesis, adaptor ligation, and universal amplification. Sequencing libraries were prepared using random priming PCR of the whole-transcriptome amplification products to enrich the 3′ end of the transcripts linked with the cell label and UMI. Sequencing libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (ThermoFisher Scientific). The libraries were sequenced on NovaSeq6000 (Illumina) using 2× 150 chemistry. The BD Rhapsody analysis pipeline was used to process raw sequencing data (fastq files). The raw sequence data have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA019550). Multiplexed immunohistochemistry Liver samples were collected, fixed in 10% formalin for 24 h and then changed to 70% ethanol before paraffin embedding; 4-μm paraffin liver sections were then prepared for histology. Multiplexed immunohistochemistry was performed standardly through adding primary antibodies sequentially, and paired with a TSA 4 color kit (M-D110061-50T, Yuanxibio). For example, from the second to fourth round of staining, slides were washed in TBST buffer and then transferred to preheated EDTA solution (100 °C) before being heat-treated using a microwave set at 20% of maximum power for 15 min. Slides were cooled in the same solution to room temperature. Incubated with anti-Human CD3 Abs (Cat#14-0037-82, eBioscience) for 60 min and then treated with horseradish peroxidase-conjugated secondary antibody (#DS9800, Leica) for 10 min. Then labeling was developed for a strictly observed 10 min, using TSA 520 per the manufacturer’s instructions. Between all steps, the slides were washed with Tris buffer. The same process was repeated for the following antibodies/fluorescent dyes, in order: anti-Human CD68 (#[61]GM351507, GeneTech)/TSA 570, anti-HBsAg Abs (#10377-MM23, Sino biological)/TSA 670, or anti-Mouse CD3e (#bsm-60002R, Bioss), anti-Mouse F4/80 (#70076, Cell Signaling Technology) or anti-Mouse CD5L Abs (#TD2304S, Abmart), and the slide was then treated with two drops of SN470 (A11010-100T; Yuanxibio), washed in distilled water, and manually coverslipped. Slides were air dried, and images taken with a Panoramic MIDI tissue imaging system (3DHISTECH). Images was analyzed using Indica Halo software. Isolation and adoptive transfer of CD3^+ macrophages Mice were anesthetized using 3% sodium pentobarbital (10 μl/g body weight) and perfused with a two-step liver perfusion protocol as described.[62]^15 After perfusion, the livers were then removed and filtered through the 100-mM mesh. The sample was suspended in HBSS and centrifuged at 50× g for 1 min at 4 °C. After collecting non-parenchymal cell-enriched supernatant, the non-parenchymal cells were purified using centrifugation with a 50%/25% two-step gradient Percoll solution after liver non-parenchymal cells were harvested, washed with HBSS containing 10% FBS and centrifuged (400× g, 5 min, 4 °C). Cells were resuspended in HBSS/FCS, counted while excluding dead cells using Trypan Blue. Liver immune cells and buffers were kept on ice during isolation protocols and subsequent analysis. Single-cell suspensions were blocked with rat anti-mouse CD16/CD32 antibodies (1:200, Cat#553142, BD Biosciences) for 15 min and afterward incubated with fluorophore-conjugated anti-mouse antibodies at recommended dilutions for 20 min at 4 °C. FACS antibodies used included: LIVE/DEAD Fixable Viability Stain700 (Cat#564997, BD Biosciences), FITC Anti-CD3(Cat#100204, Biolegend) and AlexaFluor647 anti-mouse F4/80 (Cat#123122, Biolegend), Brilliant Violet510 anti-mouse CD8a (Cat#100752, Biolegend). CD3 and F4/80 double-positive cells were isolated on a BD Aria II. For adoptive transfer experiments, 6-week-old male C57BL/6 mice were injected with pUC-B6 plasmid by hydrodynamic method to construct acute hepatitis B mice. On the third day after modeling, the sorted CD3^+ macrophages were reinfused into the mice, and each mouse was injected with approximately 5× 10^5 cells. The expression level of HBsAg in peripheral blood of mice was dynamically monitored by orbital angular vein blood collection. After 5 weeks of modeling, the sorted CD3^+ macrophages were reinfused into the slow mice. The number of reinfusion cells and blood collection methods of each mouse were the same as those of acute hepatitis B mice. CD3^+ macrophages culture supernatant inflammatory marker assays Quantitative analysis of 10 cytokines was achieved using a sandwich immunoassay (Meso Scale Diagnostics, MSD, Rockville, MD, USA). We collected the supernatant of CD3^+ macrophages after 48 h of culture, and detected interferon-gamma (IFN-γ) and IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, CXCL1, TNF-α and another 10 cytokines. Briefly, the 96-well plates were precoated with capture antibodies and the appropriate plates were blocked with 5% MSD Blocker A Solution. Calibrator dilutions were prepared, and samples were diluted using the commercial kit MSD diluents. Samples and calibrators added to the plates were incubated at room temperature with shaking for 2 h. Plates were washed thrice with a home-prepared solution of 10× PBS (pH = 7.4) (#10010001, Gibco)-Tween 20 (#A600560, Sangon Biotech). Detection antibodies were mixed with MSD diluents, incubated at room temperature, and agitated for 1–2 h. Plates were washed thrice with the PBS -Tween 20 solution, and MSD read buffer was added to the plates before examination using a SECTOR Imager 6000 (MSD). Quantitative values were then achieved using the MSD Discovery Workbench software. The coefficient of variation of the calculated values for all the biomarkers at the upper level of quantification was <20%. Myosin heavy chain-I tetramers HLA-A2–restricted HPV E7 11-19 peptide or HBV peptides, including Pol455-463 (polymerase), Core18-27 (HBcAg), Env335-343 (HBsAg), and Flex-T HLA-A∗02:01 Monomer UVX (BioLegend), were used to prepare MHC-I tetramers. Allophycocyanin (APC) H-2Kb HBsAg190–197 (VWLSVIWM) and phycoerythrin (PE) H-2Ld HBsAg28-39 (IPQSLDSWWTSL) MHC-I tetramers were purchased from HelixGen Corp. Ltd. Carboxyfluorescein diacetate succinimidyl ester -based cytotoxicity assay Six-week-old male Balb/C mice were injected with pUC-B6 plasmid using a hydrodynamic method to construct acute hepatitis B mice. The sorted CD3^+ macrophages, CD3^- macrophages, and CD8^+ lymphocytes were used as the effector cells. H2.35 mouse hepatocyte cells were plated as the target cells, These cells were cultured with HBsAg peptide (IPQSLDSWWTSL, CSBio). After washing with PBS the target cell suspensions were resuspended at 1× 10^6 cells and labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Cat#[63]C34554, ThermoFisher) for 10 min at 37 °C. Effector/target ratios of 4:1, 2:1, and 1:1 were used. The reaction was stopped by the addition of an equal volume of FCS, followed by a 2-min incubation at room temperature. After two washes the CFSE-labeled target cells were resuspended in assay medium and either directly used or cultured for 1 day at 37 °C and 5% CO[2]. The cell concentration was adjusted to 5× 10^4 cells/ml, and 100 μl/well was plated in 96-well microtiter plates. Plates were incubated in a humidified atmosphere of 5% CO[2] and 37 °C. After 4 h, relevant antibodies to stain for specific subpopulations were added and incubated at 4 °C for at least 15 min. The percentage of survival was calculated as follows: % survival = (absolute no. viable CFSE+ target cells [t = x])/(absolute no. viable CFSE+ target cells [t = 0])× 100. Statistical analysis In this study, the flow data were analyzed and exported using FlowjoV10 software, and the data after single-cell sequencing analysis provided by Bohao Company were analyzed using GraphPad Prism8 software (San Diego, CA, USA). Data are shown as mean ± SEM. Values of p were calculated using parametric or non-parametric two-way ANOVA and the paired or unpaired t test. A value of p <0.05 was considered statistically significant. Results Single-cell analysis of immune cells in the liver microenvironment of HBV mice We established HBV-persistent and fast-cleared mouse models to investigate the liver immune microenvironment during HBV infection. HBV fast-cleared mice were constructed by hydrodynamically injecting C57BL/6 mice with a 1.3-fold longer HBV-B6 genome on the pUC18 backbone,[64]^11 whereas persistent HBV-susceptible mice were generated on the basis of the AAV8-carried HBV rcccDNA model, as reported in our previous work.[65]^12 For the HBV fast-cleared mice, to compare the early stage of HBV replication and the virus clearance stage, intrahepatic non-parenchymal cells were isolated on the third day and 35th day. We applied FACS to collect CD45^+ immune cells. The viability of the cells was >90%, the concentration was 1× 10^5/ml, and the initial number of cells was 200,000. Six-week-old Alb-Cre mice were injected with 1× 10^11 copies of the AAV8-rcccDNA virus via the tail vein, as previously described.[66]^12 CD45^+ intrahepatic non-parenchymal cells were isolated on the 35th day after modeling, and mice injected with empty AAV8, pUC18, or PBS were used as controls. The basic virological indicators of these mice are shown in [67]Table S1. The mixture of CD45^+ immune cell suspensions from five mice in each model was subsequently analyzed via the BD Rhapsody™ single-cell system. The study design is briefly summarized in [68]Fig. 1A. Fig. 1. [69]Fig. 1 [70]Open in a new tab ScRNA-seq profiling of the ecosystem in HBV fast-cleared and persistent mice. (A) Overview of the experimental strategy. (B) T-distributed stochastic neighbor embedding (t-SNE) plot showing the scRNA-seq clusters of non-parenchymal cells. (C) Heatmap graph depicting feature genes in each cluster. (D) t-SNE plot showing the scRNA-seq clusters of non-parenchymal cells in separated mouse. Expression of cell-type-specific marker genes illustrated in t-SNE plots (E) and enrichment dot bubble (F). (G) Histogram indicating the cell numbers in different mice. (H) Histogram indicating the percentages of different cells from CD45^+ cells in each analyzed mouse. (I) The percentages of different cells in CD45^+ cells vary across sample origin from each analyzed mouse. scRNA-seq, single-cell RNA sequencing. Overall, 52,059 immune cell transcriptome data points were obtained, with an average number of 3,440 UMIs/cell and 1,576 genes/cell ([71]Table S2). The cells were subjected to t-distributed stochastic neighbor embedding and unsupervised clustering, which found that immune cells from all HBV mouse livers were identified into 24 clusters ([72]Fig. 1B, left), including B, macrophage, CD8^+ T, CD4^+ T, NK, granulocyte, DC, NKT, regulatory T (Treg), MAIT, and plasma cells ([73]Fig. 1B, right), which were annotated by SingleR software. The heatmap graph was used to display genes used for cell definition ([74]Fig. 1C). Their distributions in different mice are shown ([75]Fig. 1D). Levels of marker genes, including Cd79a (B and plasma), Csf1r (macrophage), Cd8a (CD8^+ T), Cd4 (CD4^+ T), Ncr1 (NK), S100a8 (granulocyte), Siglech (DC), Klrd1 (NK and NKT), Foxp3 (Treg), Zbtb16 (MAIT) and Jchain (Plasma), in immune cells are shown in [76]Fig. 1E and F. Further studies revealed that CD8^+ T cells were the most abundant population in persistent HBV-infected mice ([77]Fig. 1G–I), and could be divided into five different clusters ([78]Fig. S1A). CD8-cluster 1 expressed high levels of Xcl1, Tox, CTLA4, indicating it is exhausted population. CD8-cluster 2, defined as the effector CD8^+ T cell population, had high levels of CX3CR1, Gzma, and S1pr5. CD8-cluster 3 and 4, both have expression of Sell and Lef1 ([79]Fig. S1B), which are known as inactivated Tn- and Tmem-associated genes,[80]^16 but cluster 4 has a higher Trib2 ([81]Fig. S1B). Trib2 safeguards naive T-cell homeostasis,[82]^17 indicating cluster 4 is an earlier T cell than cluster 3. Moreover, CD8-cluster 5 expressed high levels of Pclaf, Top2a, and Mki67, suggesting it is population undergoing proliferation. Then, monocle 3 was used to analyze the dynamic immune status and cell transition trajectories of these CD8^+ T cell populations ([83]Fig. S1C). However, CD8-cluster 1 had increased PDCD1, CTLA4, TOX, HAVCR2, and CD244 expression ([84]Fig. S1D), which confirmed that it was an exhausted T cell population. The distribution of CD8 clusters in different mice was examined. The HBV-persistent (Day 35) mice had more exhausted CD8-cluster 1 cells, whereas the fast-cleared (Day 3) mice had the most abundant proliferating CD8-cluster 5 cells ([85]Fig. S1E). In contrast, macrophages greatly increased in the livers of fast-cleared mice on Days 3 and 35, and granulocytes increased on Day 3, suggesting that myeloid populations are essential to HBV clearance ([86]Fig. 1G–I). These data indicate that many CD8^+ T cells infiltrate the persistent HBV liver but lose their ability to fight the virus, whereas myeloid cells are critical populations involved in liver HBV clearance. Myeloid-derived cell components in the HBV mouse model A second-dimensional reduction was performed to further clarify the roles of myeloid populations in HBV clearance, and a total of 23,304 myeloid cells from all the mice were divided into 14 myeloid subpopulations ([87]Fig. 2A and B). The marker genes for each subpopulation are shown in violin boxes and heatmaps ([88]Fig. 2C and D). Monocyte-derived macrophages (MoMs) were divided into three subpopulations (clusters 2, 5, and 13), and tissue-resident Kupffer cells were divided into four subpopulations (clusters 0, 6, 10, and 12). The granulocytes were also divided into three subpopulations (clusters 1, 7, and 11), whereas the DCs were divided into four subpopulations: cDC-1 (cluster 3), cDC-2 (cluster 8), pDC-1 (cluster 4), and pDC-2 (cluster 9) ([89]Fig. S2A). The percentage of monocytes, granulocyte-1, and cDC-1 increased sharply on Day 3 in the HBV fast-cleared mice ([90]Fig. 2E). Furthermore, on the 35th day, pDC-1 was greater in the HBV fast-cleared model than that on Day 3 ([91]Fig. S2B), suggesting it is not a key population for viral clearance. Fig. 2. [92]Fig. 2 [93]Open in a new tab Transcriptome heterogeneity of myeloid cell clusters. (A) t-SNE showing myeloid cells vary across sample origin from each analyzed mouse. (B) t-SNE projections of granulocytes, monocyte-derived macrophage, Kupffer and DC cells, which were divided into 14 clusters from 0 to 13 in all mice. (C) Heatmap showing the scaled, average expression of typical marker genes for each of the clusters defined in myeloid-derived cells. (D) Expression of cell-type-specific marker genes illustrated by a violin map. (E) The percentages of granulocytes, monocytes, and dendritic cells subsets in myeloid cells. (F) The percentages of Kupffer-1, Kupffer-2, Kupffer-3, and Kupffer-4 in total Kupffer cells. (G) The absolute number of Kupffer subsets in mice. DC, dendritic cell; t-SNE, t-distributed stochastic neighbor embedding. Among them, macrophages were highly heterogeneous. All MoMs clusters had higher Cx3cr1, indicating they were populations migrated from periphery.[94]^18 The increased Fn1 in MoM-1 indicating that it is a subset in early differentiation from monocytes.[95]^19 High levels of C1qa in Kupffer-cluster 1–4 indicated they were tissue-resident cells.[96]^20 Kupffer-cluster 1 cells were found to be M2-like macrophages expressing high levels of APOC1 and MRC1, accumulating in the CHB liver and aiding tissue repair.[97]^21 S100a8 and S100a9-enriched Kupffer-cluster 3 were regarded as immune suppressor cells.[98]^22 Interestingly, Cd3e and Cd3g were highly expressed in Kupffer-cluster 2 (red arrow). Kupffer-cluster 4, a subpopulation with Spic gene, was present almost exclusively in the HBV fast-cleared model ([99]Fig. 2F and G), suggesting it consisted of red pulp macrophages.[100]^23 The percent of this cluster significantly increased in the livers of HBV fast-cleared and persistent mice on Day 35 ([101]Fig. 2F), but its number was only enhanced in the HBV fast-cleared mice on Day 35 ([102]Fig. 2G). Because HBV clearance in this fast-cleared model usually occurs from the third week to the fourth week after modeling,[103]^11 this cluster might be a novel macrophage population involved in eliminating HBV. Identification of intrahepatic CD3^+ Kupffer cells during HBV clearance Strikingly, in addition to Clec4f, Kupffer-cluster 2 also expressed high levels of CD3e and CD8a, which are T cell genes ([104]Fig. 3A). These cells also demonstrated increased CD3g, CD3d, Adgre1 (F4/80), and Fcgr1 (CD68) expression but not CD4 expression ([105]Fig. 3B). Nevertheless, there was a possibility that these CD3^+ macrophages were detected as a result of cells sticking. To exclude this phenomenon, we further analyzed the mitochondrial genes of the cells, which ranged from 2.5% to 5.0% in expression, suggesting they were not sticky cells. We then observed the sorted CD3^+ Kupffer cells via confocal microscopy, revealing that, similar to macrophages, they exhibited an irregular morphology and co-expressed F4/80 with CD3 and CD8 ([106]Fig. S3A). To further verify the CD3^+ Kupffer subpopulation found via scRNA-seq in the liver, the serum HBsAg of the fast-cleared mice was dynamically monitored, and the mice were sacrificed in the fifth week. Intrahepatic non-parenchymal cells were isolated and detected via flow cytometry. CD3^+ cells were gated from F4/80^high CD11b^+ Kupffer cells. As shown in [107]Fig. 3C, CD3^+ macrophages accounted for ∼8.83% of the total number of Kupffer cells. Moreover, CD3^+ F4/80^+ macrophages were observed through immunofluorescence staining of the liver ([108]Fig. 3D), confirming the scRNA-seq findings. Fig. 3. [109]Fig. 3 [110]Open in a new tab Identifying CD3^+ macrophages in HBV mice and patients. (A) t-SNE spots showing a cluster co-expressing macrophage-specific gene (Clec4f) and T cell-specific genes (CD3e, CD8a). (B) Heatmap showing the scaled average expression of T cell- and macrophage-related genes in this cluster (Kupffer-2). (C) Flow cytometry CD3^+ cells in F4/80^highCD11b^+ Kupffer cells in HBV fast-cleared mice (Day 35). (D) Immunofluorescence staining HBV fast-cleared mouse liver tissue. CD3 (green), F4/80 (red), nucleus (blue). Immunofluorescence staining CD3^+ macrophages in liver tissue from an acute patient (E), a chronic patient (F) and a chronic patient with elevated ALT (G), 63× . ALT, alanine aminotransferase; t-SNE, t-distributed stochastic neighbor embedding. We next questioned whether these CD3^+ macrophages were present in patients with hepatitis B. Liver biopsy samples from five patients with AHB and 10 patients with CHB were collected. The hepatitis B virological indicators of the two groups of patients are shown in [111]Table S3. Further H&E staining and multifluorescence labeling were performed on liver biopsy samples. H&E staining revealed that more immune cells infiltrated the liver in patients who were in acute stage rather than those who were in chronic stage ([112]Fig. S3B). CD3^+ macrophages were detected in the liver tissue of patients with AHB (n = 5, [113]Fig. 3E, [114]Fig. S3C–F) but not in those of patients with CHB (n = 10), regardless of the increase in ALT ([115]Fig. 3F and G, [116]Fig. S3G–N). These findings suggest that the number of CD3^+ macrophages increases in the liver during HBV clearance. Generation of CD3^+ macrophages via the comprehensive effects of antigen-, antibody-, and antigen-specific T cells In addition, when we checked MoMs CD11b^high and F4/80^low in B6 plasmid-based HBV fast-cleared mouse livers; CD3 and CD8 were also detected via flow cytometry ([117]Fig. S4A). Considering the above findings showing CD3^+ Kupffer cells ([118]Fig. 3C), CD3^+ macrophages likely do not constitute a certain population during development and might be generated via cell interactions. Given that these cells were found in the HBV clearance phase, we speculated that antigen-specific T cells were involved in their formulation. Recently, the roles of TCR^+ macrophages have been studied in inflammatory and infectious diseases,[119]^24 such as mycobacterial infection.[120]^25 We therefore examined HBsAg- or HBcAg-specific TCR molecules on these cells via MHC-1/peptide tetramers. The results clearly revealed that the CD3^+ macrophages carried HBsAg-specific, rather than HBcAg-specific, TCRs ([121]Fig. S4B). Next, we sorted intrahepatic CD3^+ macrophages and analyzed their TCR using a smart sequence. CD3^+ MoMs and CD3^+ Kupffer cells were separately collected from two mice. As shown in [122]Fig. S4C, the gene correlation ratios of both CD3^+ subpopulations from the two mice were close to 1, suggesting little difference between the four samples. When TCR Vα and Jα were undetectable ([123]Fig. S4D), TCR Vβ and Jβ were expressed and rearranged ([124]Fig. S4E). In addition, studies have suggested that TNF/TNFR1 expression on myeloid cells helps maintain the CD3^+ myeloid subpopulation.[125]^25^,[126]^26 Therefore, we examined the expression of TNF-related genes and found that TNF/TNFR superfamily genes, including tnfsf8, tnfrsf9, tnfrsf13c, and tnfrsf18, were specifically enriched in CD3^+ macrophages (Kupffer-cluster 2) but not in other populations ([127]Fig. S4F–I). To clarify whether CD3^+ macrophages are generated from the interaction between HBsAg-specific CD8^+ T cells and intrahepatic macrophages, we cocultured CD8^+ T cells from HBsAg TCR transgenic mice (Jackson Laboratory, No. 027510) and intrahepatic macrophages from wild-type mice. HBsAg was added to the coculture system to mimic HBV infection, which showed HBsAg alone did not induce CD3^+ macrophages ([128]Fig. 4A). Considering that these cells are present in patients with rapidly cleared or acute HBV, anti-HBsAg antibody (HBsAb) was added. The results showed that the combination of HBsAg and HBsAb could efficiently induce CD3^+ macrophages in the presence of HBsAg T cell receptor transgenic (TCRtg) CD8^+ T cells ([129]Fig. 4A). In the absence of T cells, macrophages did not change to the CD3^+ phenotype after the addition of HBsAg and/or HBsAb ([130]Fig. 4B). We then replaced TCRtg T cells with wild-type CD8^+ T cells. The CD3^+F4/80^+ population also increased but was significantly lower than that in the TCRtg T cell group ([131]Fig. 4C). Another question is whether CD3^+ macrophages are derived from T cells or macrophages, as enrichment of CD14^-positive CD8^+ T cells has been demonstrated in the liver.[132]^27 Thus, we performed a transwell assay, and CD3^+F4/80^+ cells were found in the lower chamber seeded with macrophages but not in the upper insert containing T cells ([133]Fig. 4D). Moreover, HBsAg and HBsAb dose-dependently induced these CD3^+ macrophages ([134]Fig. 4E). This process was also dependent on the combination of HBsAg and HBsAb, whereas the addition of HBsAg alone had no effect ([135]Fig. 4F). To further evaluate the generation of CD3^+ macrophages in vivo, we constructed HBV fast-cleared models in Rag1 knockout (lack of mature T and B cells), thymus-deficient nude (lack of mature T cells), and muMt mice who are deficient in membrane-bound IgM and lack of mature B cells. The mice were sacrificed between weeks 4 and 5 after hydrodynamic injection of the HBV B6 plasmid. Liver tissues were stained with F4/80 and CD3 antibodies. CD3 and F4/80 double-positive cells were observed in wild-type mice ([136]Fig. 4G) but not in livers from Rag1 knockout, thymus-deficient nude, or muMt knockout mice ([137]Fig. 4H–J). These data indicate that CD3^+ macrophages originate from intrahepatic macrophages and are produced via the cooperated effects of antigen-, antibody-, and antigen-specific T cells. Fig. 4. [138]Fig. 4 [139]Open in a new tab Investigating the origin of CD3^+ macrophages. (A) Inducing CD3^+F4/80^+ cells in coculture of liver macrophages and HBsAg-specific CD8^+ T cells in the presence of HBsAg and/or HBsAb 5 μg/ml (n = 6), respectively. (B) Stimulating macrophages with HBsAg and/or HBsAb without T cells (n = 3). (C) Stimulating macrophages with HBsAg and/or HBsAb in the present of wild-type (WT) or TCR transgeinic (TCRtg) T cells. (D) Transwell culture liver macrophages and HBsAg-specific CD8^+ T cells (n = 5). (E) Inducing CD8^+ macrophages in HBsAb-added transwell culture system in a dose-dependent manner by HBsAg. (F) Stimulating macrophages with dose-dependent HBsAg, without HBsAb, in transwell coculture with T cells. Detection of F4/80 and CD3 double-positive cells in livers from WT (G), Rag1 knockout (H), nude (I) and muMt knockout mice (J) on Week 4–5 after modeling, 100× . Parametric t test was used. ∗∗p <0.01 and ∗∗∗p <0.001. CD3^+ macrophages are characterized by long-term residence in the liver Next, we investigated whether CD3^+ macrophages disappear after HBV clearance. Six-week-old C57BL/6 mice were selected to construct an HBV fast-cleared mouse model, and the number of CD3^+ macrophages in the livers of the mice was dynamically monitored at 2, 3, 5, 8, and 12 weeks after modeling. From the third week, as the HBsAg level decreased ([140]Fig. 5A), the proportion of CD3^+ macrophages peaked (0.4%). The proportion of this subpopulation decreased slightly in the fifth week ([141]Fig. 5B). However, before the HBsAg level decreased, the number of CD3^+ Kupffer cells in these fast-cleared mice remained low during the second week after modeling ([142]Fig. 5B). Interestingly, CD3^+ macrophages persisted until 12 weeks ([143]Fig. 5B), although the ALT had declined since the fifth week ([144]Fig. 5C). The dynamic changes in CD3^+ macrophages in the livers of the mice were also confirmed via confocal microscopy. CD3^+ CDF4/80^+ double-positive macrophages were observed from the third to the eighth week ([145]Fig. 5D). Fig. 5. [146]Fig. 5 [147]Open in a new tab CD3^+ macrophages are characterized by long-term residing in liver with enhanced CD5L. (A) Detecting serum HBsAg in these mice (n = 7). (B) Dynamically monitoring intrahepatic CD3^+ macrophages in HBV fast-cleared mice (B6) after modeling by flow cytometry (n = 4). (C) Dynamically monitoring serum ALT levels in HBV fast-cleared mice (n=4). (D) Immunofluorescence staining intrahepatic CD3^+ macrophages from mice, 100×. (E) Immunofluorescence staining CD5L in intrahepatic CD3^+ macrophages from HBV fast-cleared mice, 63× . (F) Immunofluorescence staining CD5L in intrahepatic CD3^+ macrophages from acute hepatitis B patients, 63×. Two-way ANOVA was used. ∗∗∗p <0.001. We then analyzed scRNA-seq data to determine the level of CD5L, a known inhibitor of macrophage apoptosis, and found that CD5L expression was significantly upregulated in CD3^+ macrophages in scRNA-seq figures (Kupffer-cluster 2, [148]Fig. 2C, green arrow). The CD5L protein was also found to colocalize with CD3 and F4/80 in mouse liver tissue ([149]Fig. 5E), which was confirmed in tissue from liver biopsies of acute hepatitis B patients ([150]Fig. 5F). Moreover, increased expression of Bcl2, typically considered an apoptosis inhibitor in T cells, was observed via scRNA-seq ([151]Fig. S5A) and colocalized with CD3 and F4/80 in both mouse and patient liver tissues ([152]Figs. S5B and C). This finding suggested that CD3^+ macrophages are long-term resident cells that fight viral infection. CD3^+ macrophages are a polyfunctional population against HBV The function of CD3^+ macrophages is very interesting. The enrichment analysis data from scRNA-seq via gene set enrichment analysis revealed that the Notch, VEGF, and interferon-gamma signaling pathways were activated in CD3^+ macrophages ([153]Fig. 6A). CD3^+ macrophages were selected and cultured to express proinflammatory activation-related cytokines. Then, the supernatants were collected, and 10 cytokines (IFN-γ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, CXCL1, and TNF-α) were detected. Moreover, CD206^+ macrophages sorted from the livers of mice were used as M2 tissue repair macrophages, and CD206^- macrophages were used as the M1 proinflammatory activated macrophage subpopulation for comparison. As shown in [154]Fig. 6B, CD3^+ macrophages highly expressed CXCL1 (257.84 pg/ml), which was significantly different from CD3^- macrophages, CD206^+ macrophages and CD206^- macrophages. The results of the transcription analysis ([155]Fig. S4G–I) revealed that the level of TNF-α was significantly high (101.86 pg/ml) in the culture supernatant of the CD3^+ macrophages ([156]Fig. 6C). In addition, the IL-6 level was also high, with a mean value of 172.09 pg/ml, but was lower than that of CD206^- macrophages ([157]Fig. 6D). CD3^+ macrophages did not show any advantage in the secretion of IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12, or IFN-γ ([158]Figs. S6A–D). Fig. 6. [159]Fig. 6 [160]Open in a new tab Functions of CD3^+ macrophages in against HBV. (A) GSEA signaling pathway enrichment analysis of CD3^+ macrophages (n = 12), CD3^- macrophages (n = 17), CD206^- macrophages (n = 13), and CD206^+ macrophages (n = 12). Evaluating CXCL1 (B), TNF-α (C), and IL-6 (D) in the culture supernatants of macrophages. (E) Analyzing phagocytic ability of macrophages by engulfing phycoerythrin (PE)-labeled fluorescent microspheres. (F) Comparing the phagocytic ability of CD3^+ and CD3^- macrophages. (G) Coculture macrophages (effector, E) with peptide-loaded hepatocytes (target, T), detecting death ratio of targeted hepatocytes. (H, I) Comparing the cytotoxic capability of CD3^+ macrophages with CD8^+ T cells. (J) Monitoring serum HBsAg in HBV fast-cleared HBV model mice after adoptive transfer of CD3^+ or CD3^- macrophages (5× 10^5 per mouse), n = 5, respectively. Non-parametric t test was used. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, and ∗∗∗∗p<0.0001. GSEA, gene set-enrichment analysis. Because these cells are essentially a macrophage population, we checked their phagocytic function. CD3^+ and CD3^- macrophages were sorted to study their ability to engulf PE-labeled fluorescent microspheres. Although TCRβ^+ macrophages have an enhanced phagocytic ability,[161]^28 our flow cytometry results revealed that both the CD3^+ and CD3^- macrophage subpopulations could engulf microspheres efficiently without distinction ([162]Fig. 6E and F). Because these cells also express lymphocyte surface molecules, CD3^+ macrophages and CD8^+ T cells in the livers of HBV fast-cleared BALB/c mice (B6 plasmid-based) were sorted for killing experiments, and the peptide-loaded H2.35 liver cell line (BALB/c background) was used as the target cell line. CD3^+ macrophages effectively killed the target cells in a dose-dependent manner ([163]Fig. 6G). Then, we compared the cytotoxic effects of CD3^+ macrophages and CD8^+ T cells (E:T = 2:1) and found that CD3^+ macrophages efficiently killed targets but were much weaker than CD8^+ T cells ([164]Fig. 6H and I). Next, we studied whether CD3^+ macrophages could promote HBV clearance in vivo. These cells were isolated from HBV fast-cleared mice and adoptively transferred into HBV mice. The number of adopted cells was 5× 10^5 for each mouse. Blood samples were collected at different time points to dynamically observe the changes in HBsAg in plasma. HBsAg levels decreased quickly in mice that received CD3^+ macrophages, and HBsAg was cleared on Day 7 ([165]Fig. 6J). Conversely, compared with untransfected mock-treated mice, mice that received CD3^- macrophages did not show accelerated HBsAg clearance ([166]Fig. 6J). These findings suggest that CD3^+ macrophages promote HBV clearance in vivo. Discussion HBV infection can result in AHB or CHB; however, the mechanism involved in the distinct outcomes is incompletely understood. Our previous work revealed that HBsAg-carrying monocytic myeloid-derived suppressor cells could promote HBV-specific T-cell tolerance after migrating into the thymus.[167]^4 Nevertheless, the type of immune cell that contributes to HBV clearance in acute and self-limited hepatitis B in the liver needs further research. In this study, we compared the liver immune environments of HBV fast-cleared and persistent mice via scRNA-seq. We revealed that intrahepatic myeloid populations were predominant in HBV-cleared mice. In addition, we identified polyfunctional CD3^+ macrophages that could help eliminate HBV and reside in the liver at least 10 weeks after the loss of HBsAg. Interestingly, these cells are generated through interactions between macrophages and antigen-specific CD8^+ T cells in the presence of HBsAg and HBsAb, indicating that macrophages and T/B cells are important for HBV clearance. ScRNA-seq technology is used for transcriptome studies on immune cells in disease-related microenvironments. Through powerful bioinformatics analysis, the immune-ecological environment at the single-cell level can be comprehensively analyzed, the heterogeneity of the immune response process and immune tolerance can be analyzed, and new immune subpopulations and markers can be discovered. Liver-resident immune cells have been studied for their heterogeneity, and CXCR6^+ T and NK cells are considered preferential populations in healthy livers.[168]^29 Importantly, similar to our findings in persistent HBV-infected mice, CD8^+ T cells have been shown via scRNA-seq to be exhausted in clinical patients with CHB and acutely recovered hepatitis B donors.[169]^30 However, macrophages constitute one of the predominant cell types in the liver. ScRNA-seq has been used to identify distinct intrahepatic macrophage subpopulations[170]^31 and clarify their roles in non-alcoholic fatty liver disease.[171]^32 Thus, the heterogeneity of intrahepatic macrophages in hepatitis B must be clarified. In this work, we performed scRNA-seq and observed seven monocyte and macrophage subpopulations in HBV model mice, including three monocyte and four Kupffer cell populations. Kupffer-2, characterized by the expression of T cell genes, was dramatically enriched in the liver during HBV clearance, and adoptive transfer accelerated the elimination of HBV in vivo. These CD3^+ macrophages were also observed in liver biopsy tissues from patients with AHB but not in tissues from patients with CHB, suggesting that they are crucial populations against HBV infection. T cells play central roles in eliminating HBV. Vigorous and polyclonal virus-specific CD8^+ T cells have been observed in acute and self-limited hepatitis B patients.[172]^33^,[173]^34 CD8^+ T-cell deletion promotes viral persistence in naturally HBV-infected chimpanzees.[174]^35 Moreover, infiltrated monocyte-derived macrophages defend against HBV.[175]^36 A subset of Kupffer cells that respond to IL-2 can also efficiently increase the number of intrahepatic HBV-specific CD8^+ T cells,[176]^37 indicating that the interaction between macrophages and T cells is powerful in HBV defense. Our study revealed that CD3^+ macrophages expressed T cell genes and exhibited phagocytic and cytotoxic capabilities. Notably, these cells could be derived from both infiltrated macrophages and resident Kupffer cells, suggesting that cell interactions constitute the mechanism involved in their generation. Recently, a study revealed that CD14^-positive CD8^+ T cells are compartmentalized in the human liver and acquire enhanced antiviral effector function during HBV infection.[177]^27 In contrast, the CD3^+F4/80^+ cells identified here are macrophages rather than T cells, as carefully demonstrated through transwell culture. In addition, coculture of HBsAg-specific T cells with intrahepatic macrophages verified that HBsAg and HBsAb were indispensable for the generation of CD3^+ macrophages, which was further demonstrated by establishing a fast-cleared HBV model in T-cell- and/or B-cell-deficient mice. Also, M1 and M2 macrophages play distinct and crucial roles in the liver. Interestingly, the CD3^+ macrophages identified in this study have enhanced TNF signaling, suggesting them like to be M1 phenotype. What we hope to explain is that M1/M2 is a classification method for macrophages, CD3^+ macrophage may not classically be defined as M1 or M2, because they showed unique effects. Correspondingly, several studies have demonstrated that TNF signaling can regulate the generation of TCR^+ macrophages and CD3^+ myeloid cells.[178]^25^,[179]^26^,[180]^38 Accordingly, our findings revealed that TNF-α expression increased in the culture supernatant of CD3^+ macrophages and that the transcription of tnfsf8, tnfrsf9, tnfrsf13c, and tnfrsf18 increased. It is possible that TNF signaling is activated by antigen-specific CD8+ T cells. The detail mechanism remains further study. Recently, an increasing number of studies have shown that protective processes and actions during secondary infection are related to B and T cells and innate immune cells, such as NK cells and macrophages. This process, referred to as trained innate immunity, is formulated via metabolic alterations and epigenetic modifications.[181]^39^,[182]^40 Strikingly, here, we observed that CD3^+ macrophages persist in the mouse liver for over 10 weeks after the loss of HBsAg. Despite the lack of direct evidence, we strongly speculate that these long-term resident CD3^+ macrophages are a form of trained innate immunity, which needs further investigation. Similar to our findings that long-term residing CD3^+ macrophages originate from both monocyte-derived macrophages and Kupffer cells, Bacillus Calmette–Guerin (BCG) is an inducer of trained immunity in monocytes and drives the long-term activation of lung-resident macrophages, restricting Mycobacterium tuberculosis replication.[183]^41^,[184]^42 We also found that these CD3^+ macrophages express high levels of Bcl2 and CD5L. Bcl2 usually acts as an apoptosis regulator in T cells,[185]^43 and increased Bcl2 might protect CD3^+ macrophages from apoptosis. CD5L is expressed primarily by macrophages[186]^44 and influences macrophage activation and plasticity.[187]^45^,[188]^46 Notably, CD5L is an efficient inhibitor of macrophage apoptosis.[189]^47^,[190]^48 A limitation is that liver tissue from patients was used to confirm it was a long-term resident population, as it is almost impossible to undergo biopsy after HBV cure. Our evidence indicates that CD3^+ macrophages, which are involved in HBV clearance, acquire resistance to apoptosis and might be activated quickly during the next infection. Moreover, in contrast to trained innate immunity, HBsAg-specific TCRs are detectable on intrahepatic CD3^+ macrophages, allowing them to respond to HBV selectively. The TCR is usually restricted to T cells; however, consistent with our findings, several studies have shown that it is detectable on macrophages.[191]^24^,[192]^49 αβTCR-positive macrophages have been identified in human and murine malignancies and HPV-related head and neck squamous cell carcinoma.[193]^49^,[194]^50 Another study demonstrated that CD3^+TCR^+ macrophages are differentiated from monocytes during BCG infection.[195]^38 Our data also clearly revealed that TCR-expressing intrahepatic CD3^+ macrophages kill peptide-loaded hepatocytes. The results revealed the function of the TCR on CD3^+ macrophages in recognizing complexes of MHC-1 and epitopes, although their cytotoxicity was weaker than that of CD8^+ T cells. In conclusion, we provide insights into the liver immune microenvironment in HBV-exposed mice using scRNA-seq, revealing that long-term residing CD3^+ macrophages perform phagocytosis and cytotoxic functions. These findings could help elucidate the role of intrahepatic immune responses in eliminating HBV and establish a potential therapeutic strategy for CHB. Abbreviations AAV8, adeno-associated virus 8; AHB, acute hepatitis B; ALT, alanine aminotransferase; CFSE, carboxyfluorescein diacetate succinimidyl ester; CHB, chronic hepatitis B; DC, dendritic cell; GSEA, gene set enrichment analysis; HBsAb, anti-HBsAg antibody; HSCs, hepatic stellate cells; IFN-γ, interferon-gamma; LSEC, liver sinusoidal endothelial cells; MAIT, mucosal-associated invariant T; MoM, monocyte-derived macrophages; NK, natural killer; NKT cell, natural killer T cell; rcccDNA, recombinant covalently closed circular DNA; scRNA-seq, single-cell RNA sequencing; TCR, T cell receptor; TNF-α, tumor necrosis factor alpha; Treg cell, regulatory T cell; t-SNE, t-distributed stochastic neighbor embedding; UMI, unique molecular identifier. Financial support This study was funded by National Key R&D Program of China (No. 2021YFC2300602 to ZY), the National Natural Science Foundation of China (No. U23A20474 and 91842309 to ZY), the Shanghai Municipal Science and Technology Major Project (ZD2021CY001 to ZY).‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬ Authors’ contributions Designed the experiments: ZY, ZF. Prepared the figures and/or wrote the manuscript: CW, ZF. Performed and/or analyzed ex vivo experiments: CW, WZ, KZ, BS, MW. Performed and/or analyzed in vivo experiments: CW, ZF, XZ. Gave suggestions regarding experiments: MK, XZ. Data availability statement The raw sequence data have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA019550). Conflicts of interest The authors declare no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details. Footnotes Author names in bold designate shared co-first authorship Supplementary data to this article can be found online at [196]https://doi.org/10.1016/j.jhepr.2024.101323. Contributor Information Zhong Fang, Email: zhongfang13@fudan.edu.cn. Zhenghong Yuan, Email: zhyuan@shmu.edu.cn. Supplementary data The following is the Supplementary data to this article: Multimedia component 1 [197]mmc1.pdf^ (1.6MB, pdf) Multimedia component 2 [198]mmc2.docx^ (40.6KB, docx) Multimedia component 3 [199]mmc3.pdf^ (1.7MB, pdf) Multimedia component 4 [200]mmc4.pdf^ (6.1MB, pdf) References