RESUMO
Elucidating the cellular organization of the cerebral cortex is critical for understanding brain structure and function. Using large-scale single-nucleus RNA sequencing and spatial transcriptomic analysis of 143 macaque cortical regions, we obtained a comprehensive atlas of 264 transcriptome-defined cortical cell types and mapped their spatial distribution across the entire cortex. We characterized the cortical layer and region preferences of glutamatergic, GABAergic, and non-neuronal cell types, as well as regional differences in cell-type composition and neighborhood complexity. Notably, we discovered a relationship between the regional distribution of various cell types and the region's hierarchical level in the visual and somatosensory systems. Cross-species comparison of transcriptomic data from human, macaque, and mouse cortices further revealed primate-specific cell types that are enriched in layer 4, with their marker genes expressed in a region-dependent manner. Our data provide a cellular and molecular basis for understanding the evolution, development, aging, and pathogenesis of the primate brain.
Assuntos
Córtex Cerebral , Macaca , Análise de Célula Única , Transcriptoma , Animais , Humanos , Camundongos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Macaca/metabolismo , Transcriptoma/genéticaRESUMO
Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.
Assuntos
Neurogênese/fisiologia , Neuroglia/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Sistemas CRISPR-Cas/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Modelos Animais de Doenças , Dopamina/metabolismo , Regulação da Expressão Gênica/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças do Sistema Nervoso/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Células Ganglionares da Retina/fisiologiaRESUMO
CRISPR-based genome editing holds promise for genome engineering and other applications in diverse organisms. Defining and improving the genome-wide and transcriptome-wide specificities of these editing tools are essential for realizing their full potential in basic research and biomedical therapeutics. This review provides an overview of CRISPR-based DNA- and RNA-editing technologies, methods to quantify their specificities, and key solutions to reduce off-target effects for research and improve therapeutic applications.
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Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , DNA/genética , Genoma/genética , TranscriptomaRESUMO
Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA12, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.
Assuntos
DNA/genética , Edição de Genes/métodos , Mutagênese , Mutação , Nucleosídeo Desaminases/genética , Engenharia de Proteínas , RNA/genética , Adenina/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Citosina/metabolismo , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Células HEK293 , Humanos , Nucleosídeo Desaminases/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUE-S at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE-S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUE-S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.
Assuntos
Citidina Desaminase/genética , Proteínas/genética , Edição de RNA , Núcleo Celular/metabolismo , Células HEK293 , Humanos , RNA/química , RNA/metabolismo , RNA Guia de Cinetoplastídeos , TranscriptomaRESUMO
Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant-YE1-BE3-FNLS-that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits.
Assuntos
Proteína 9 Associada à CRISPR/genética , Citosina/metabolismo , DNA/genética , Edição de Genes/métodos , RNA/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Células HEK293 , Humanos , Mutação , Mutação PuntualRESUMO
BACKGROUND: Personalized therapy has been at the forefront of cancer care, making cancer treatment more effective. Since cancer patients respond individually to drug therapy, predicting the sensitivity of each patient to specific drugs is very helpful to apply therapeutic agents. Traditional methods focus on node (molecular) information but ignore relevant interactions among different nodes, which has very limited application in complex situations, such as cancer drug responses in real clinical practice. RESULTS: Treatment evaluation with Quantified Network (TreeQNet) is a webserver which could predict sensitivity to drugs for patients through the innovative use of proteomic and phosphoproteomic network from tumor tissues. CONCLUSION: TreeQNet service: http://bioinfo.ustc.edu.cn/ . TreeQNet source code: https://github.com/Really00/treeqnet-web-front/ .
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Antineoplásicos , Neoplasias , Humanos , Proteômica , Software , Neoplasias/tratamento farmacológicoRESUMO
In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Zigoto/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Sequência de Bases , Cóclea/metabolismo , Surdez/genética , Surdez/fisiopatologia , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Motores Moleculares/metabolismo , Mutação/genéticaRESUMO
Damage or degeneration of inner ear spiral ganglion neurons (SGNs) causes hearing impairment. Previous in vitro studies indicate that cochlear glial cells can be reprogrammed into SGNs, however, it remains unknown whether this can occur in vivo. Here, we show that neonatal glial cells can be converted, in vivo, into SGNs (defined as new SGNs) by simultaneous induction of Neurog1 (Ngn1) and Neurod1. New SGNs express SGN markers, Tuj1, Map2, Prox1, Mafb and Gata3, and reduce glial cell marker Sox10 and Scn7a. The heterogeneity within new SGNs is illustrated by immunostaining and transcriptomic assays. Transcriptomes analysis indicates that well reprogrammed SGNs are similar to type I SGNs. In addition, reprogramming efficiency is positively correlated with the dosage of Ngn1 and Neurod1, but declined with aging. Taken together, our in vivo data demonstrates the plasticity of cochlear neonatal glial cells and the capacity of Ngn1 and Neurod1 to reprogram glial cells into SGNs. Looking ahead, we expect that combination of Neurog1 and Neurod1 along with other factors will further boost the percentage of fully converted (Mafb+/Gata3+) new SGNs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/metabolismo , Animais , Sequência de Bases , Imunofluorescência , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MafB/genética , Fator de Transcrição MafB/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Clathrin-mediated endocytosis is an essential cellular process that involves the concerted assembly and disassembly of many different proteins at the plasma membrane. In yeast, live-cell imaging has shown that the spatiotemporal dynamics of these proteins is highly stereotypical. Recent work has focused on determining how the timing and functions of endocytic proteins are regulated. In this Cell Science at a Glance article and accompanying poster, we review our current knowledge of the timeline of endocytic site maturation and discuss recent works focusing on how phosphorylation, ubiquitylation and lipids regulate various aspects of the process.
Assuntos
Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/fisiologia , Animais , Humanos , Metabolismo dos Lipídeos , Fosforilação , UbiquitinaçãoAssuntos
Epilepsia , Convulsões , Camundongos , Animais , Convulsões/genética , Convulsões/terapia , Epilepsia/genética , Epilepsia/terapia , FenótipoRESUMO
Anionic phospholipids PI(4,5)P2 and phosphatidylserine (PS) are enriched in the cytosolic leaflet of the plasma membrane where endocytic sites form. In this study, we investigated the roles of PI(4,5)P2 and PS in clathrin-mediated endocytosis (CME) site initiation and vesicle formation in Saccharomyces cerevisiae. Live-cell imaging of endocytic protein dynamics in an mss4(ts) mutant, which has severely reduced PI(4,5)P2 levels, revealed that PI(4,5)P2 is required for endocytic membrane invagination but is less important for endocytic site initiation. We also demonstrated that, in various deletion mutants of genes encoding components of the Rcy1-Ypt31/32 GTPase pathway, endocytic proteins dynamically assemble not only on the plasma membrane but also on intracellular membrane compartments, which are likely derived from early endosomes. In rcy1Δ cells, fluorescent biosensors indicated that PI(4,5)P2 only localized to the plasma membrane while PS localized to both the plasma membrane and intracellular membranes. Furthermore, we found that polarized endocytic patch establishment is defective in the PS-deficient cho1Δ mutant. We propose that PS is important for directing endocytic proteins to the plasma membrane and that PI(4,5)P2 is required to facilitate endocytic membrane invagination.
Assuntos
Clatrina/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Endocitose , Proteínas F-Box/metabolismo , Membranas Intracelulares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.
Assuntos
Endocitose , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Membrana/química , Dados de Sequência Molecular , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Biochar exhibits numerous advantages in enhancing the soil environment despite a few limitations due to its lower surface energy. Nanomodified biochar combines the advantages of biochar and nanoscale materials. However, its effects on water infiltration and N leaching in a clayey soil remain unclear. Therefore, this study prepared rice straw nano-biochar by a ball milling method, and investigated its physicochemical properties and effects of bulk biochar and nano-biochar at various addition rates (0 %, 0.5 %, 1 %, 2 %, 3 %, and 5 %) on wetting peak migration, cumulative infiltration, water absorption and retention, and N leaching. The results showed that, compared with bulk biochar, nano-biochar presented a more abundant pore structure with an increase in specific surface area of approximately 1.5 times, accompanied by a 20 % increase in acid functional groups. Compared with those for clayey soil without biochar addition, the wetting front migration time was increased by 10.2 %-123.9 % and 17.0 %-257.9 %, and the cumulative infiltration volume at 60 min was decreased by 26.0 %-48.4 % and 14.1 %-62.4 % for bulk biochar and nano-biochar, respectively. The parameter S of Philip model and the parameter a of Kostiakov model for nano-biochar were lower than those for bulk biochar, whereas the parameter b of Kostiakov model was greater, indicating that nano-biochar decreased initial soil infiltration rate and increased attenuation degree of the infiltration rate. Nano-biochar increased water absorption by 8.03 % and subsequently enhanced water retention capacity relative to bulk biochar. In addition, bulk biochar and nano-biochar reduced NH4+-N leaching by 3.0 %-13.1 % and 5.7 %-39.2 %, respectively, and NO3--N leaching by 2.7 %-3.6 % and 9.0 %-43.3 %, respectively, by decreasing N concentration and leachate volume relative to those with no biochar addition. This study provides new knowledge for nano-biochar application in a clayey soil.
Assuntos
Carvão Vegetal , Argila , Nitrogênio , Oryza , Solo , Carvão Vegetal/química , Solo/química , Nitrogênio/análise , Argila/química , Poluentes do Solo/análise , Água/químicaRESUMO
To improve the retention and slow-release abilities of nitrogen (N) and phosphorus (P), an 82 %-purity struvite fertilizer (MAP-BC) was synthesized using magnesium-modified biochar and a solution with a 2:1 concentration ratio of NH4+ to PO43- at a pH of 8. Batch microscopic characterizations and soil column leaching experiments were conducted to study the retention and slow-release mechanisms and desorption kinetics of MAP-BC. The slow-release mechanism revealed that the dissolution rate of high-purity struvite was the dominant factor of NP slow release. The re-adsorption of NH4+ and PO43- by biochar and unconsumed MgO prolonged slow release. Mg2+ ionized by MgO could react with PO43- released from struvite to form Mg3(PO4)2. The internal biochar exhibited electrostatic attraction and pore restriction towards NH4+, while magnesium modification and nutrient loading formed a physical antioxidant barrier that ensured long-term release. The water diffusion experiment showed a higher cumulative release rate for PO43- compared to NH4+, whereas in soil column leaching, the trend was reversed, suggesting that soil's competitive adsorption facilitated the desorption of NH4+ from MAP-BC. During soil leaching, cumulative release rates of NH4+ and PO43- from chemical fertilizers were 3.55-3.62 times faster than those from MAP-BC. The dynamic test data for NH4+ and PO43- in MAP-BC fitted the Ritger-Peppas model best, predicting release periods of 163 days and 166 days, respectively. The leaching performances showed that MAP-BC reduced leaching solution volume by 5.58 % and significantly increased soil large aggregates content larger than 0.25 mm by 24.25 %. The soil nutrients retention and pH regulation by MAP-BC reduced leaching concentrations of NP. Furthermore, MAP-BC significantly enhanced plant growth, and it is more suitable as a NP source for long-term crops. Therefore, MAP-BC is expected to function as a long-term and slow-release fertilizer with the potential to minimize NP nutrient loss and replace part of quick-acting fertilizer.
Assuntos
Fertilizantes , Magnésio , Estruvita/química , Magnésio/química , Fertilizantes/análise , Óxido de Magnésio , Fósforo/química , Carvão Vegetal/química , Solo/química , Nitrogênio/análiseRESUMO
In recent years, the application of single-cell transcriptomics and spatial transcriptomics analysis techniques has become increasingly widespread. Whether dealing with single-cell transcriptomic or spatial transcriptomic data, dimensionality reduction and clustering are indispensable. Both single-cell and spatial transcriptomic data are often high-dimensional, making the analysis and visualization of such data challenging. Through dimensionality reduction, it becomes possible to visualize the data in a lower-dimensional space, allowing for the observation of relationships and differences between cell subpopulations. Clustering enables the grouping of similar cells into the same cluster, aiding in the identification of distinct cell subpopulations and revealing cellular diversity, providing guidance for downstream analyses. In this review, we systematically summarized the most widely recognized algorithms employed for the dimensionality reduction and clustering analysis of single-cell transcriptomic and spatial transcriptomic data. This endeavor provides valuable insights and ideas that can contribute to the development of novel tools in this rapidly evolving field.
RESUMO
Bone metastasis is of common occurrence in renal cell carcinoma with poor prognosis, but no optimal treatment approach has been established for bone metastatic renal cell carcinoma. To explore the potential therapeutic targets for bone metastatic renal cell carcinoma, we profile single cell transcriptomes of 6 primary renal cell carcinoma and 9 bone metastatic renal cell carcinoma. We also include scRNA-seq data of early-stage renal cell carcinoma, late-stage renal cell carcinoma, normal kidneys and healthy bone marrow samples in the study to better understand the bone metastasis niche. The molecular properties and dynamic changes of major cell lineages in bone metastatic environment of renal cell carcinoma are characterized. Bone metastatic renal cell carcinoma is associated with multifaceted immune deficiency together with cancer-associated fibroblasts, specifically appearance of macrophages exhibiting malignant and pro-angiogenic features. We also reveal the dominance of immune inhibitory T cells in the bone metastatic renal cell carcinoma which can be partially restored by the treatment. Trajectory analysis showes that myeloid-derived suppressor cells are progenitors of macrophages in the bone metastatic renal cell carcinoma while monocytes are their progenitors in primary tumors and healthy bone marrows. Additionally, the infiltration of immune inhibitory CD47+ T cells is observed in bone metastatic tumors, which may be a result of reduced phagocytosis by SIRPA-expressing macrophages in the bone microenvironment. Together, our results provide a systematic view of various cell types in bone metastatic renal cell carcinoma and suggest avenues for therapeutic solutions.
Assuntos
Neoplasias Ósseas , Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Neoplasias Ósseas/genética , Macrófagos/metabolismo , Microambiente TumoralRESUMO
Adenine base editors (ABEs) and cytosine base editors (CBEs) enable the single nucleotide editing of targeted DNA sites avoiding generation of double strand breaks, however, the genomic features that influence the outcomes of base editing in vivo still remain to be characterized. High-throughput datasets from lentiviral integrated libraries were used to investigate the sequence features affecting base editing outcomes, but the effects of endogenous factors beyond the DNA sequences are still largely unknown. Here the base editing outcomes of ABE and CBE were evaluated in mammalian cells for 5012 endogenous genomic sites and 11,868 genome-integrated target sequences, with 4654 genomic sites sharing the same target sequences. The comparative analyses revealed that the editing outcomes of ABE and CBE at endogenous sites were substantially different from those obtained using genome-integrated sequences. We found that the base editing efficiency at endogenous target sites of both ABE and CBE was influenced by endogenous factors, including epigenetic modifications and transcriptional activity. A deep-learning algorithm referred as BE_Endo, was developed based on the endogenous factors and sequence information from our genomic datasets, and it yielded unprecedented accuracy in predicting the base editing outcomes. These findings along with the developed computational algorithms may facilitate future application of BEs for scientific research and clinical gene therapy.
RESUMO
Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB+ and CD8 proliferating proportions and a low Macro CD5L+ proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB+ and CD8 proliferating proportions are increased, but the CD8 GZMK+ proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB+ to CD8 GZMK+ facilitates good response to the therapy, while Macro CD5L+-CD8 GZMB+ crosstalk impairs the response by increasing CTLA4 in CD8 GZMB+. Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8+T-cell status conversion and exhaustion induced by Macro CD5L+ in influencing the response, suggesting future avenues for cancer treatment optimization.