OBJECTIVES: To investigate the clinical significance of SLC1A5 overexpression in pan-cancer and its mechanism for promoting hepatocellular carcinoma (HCC) progression. METHODS: We analyzed the correlation of SLC1A5 expression with clinical stage, lymph node metastasis and prognosis in pan-cancer using TCGA and ICGC datasets and explored its association with immune cell infiltration using EPIC, CIBERSORT, and TIMER algorithms. In HCC cell lines, the effects of lentivirus-mediated SLC1A5 overexpression or RNA interference on cell proliferation were examined using CCK-8 assay, and the growth of HCC cell xenografts overexpressing SLC1A5 was observed in nude mice. The effects of SLC1A5 overexpression or silencing in HCC cells on macrophage polarization were evaluated in a cell co-culture system. RESULTS: SLC1A5 was mainly localized on cell membrane and was highly expressed in most cancers in association with clinical stage, lymph node metastasis and poor prognosis. SLC1A5 expression was positively correlated with immunity score in 13 cancer types, especially in low-grade glioma (LGG), LIHC and thyroid cancer. SLC1A5 was positively correlated with macrophage infiltration level in LGG and LIHC but negatively correlated with macrophage infiltration in 5 cancers including lung squamous carcinoma, pancreatic carcinoma, and gastric carcinoma. Patients with SLC1A5 overexpression and high level of M2 macrophage infiltration had the worst survival outcomes. SLC1A5 was correlated with immunosuppression-related genes, cytokines, and cytokine receptors, which was the most obvious in LGG and LIHC. SLC1A5 was highly expressed in different HCC cell lines, and its overexpression promoted HCC cell proliferation both in vitro and in nude mice. In the cell co-culture experiment, SLC1A5 was positively correlated with the molecular markers of M2 polarization of macrophages, and its overexpression strongly promoted M2 polarization of the macrophages and inhibited T cell secretion of IFN-γ. CONCLUSIONS SLC1A5 expression level is correlated with clinical stage, lymph node metastasis, prognosis, and immune cell infiltration in most cancers, and its overexpression promotes HCC progression by inhibiting T-cell function via promoting M2 polarization of macrophages.
Humans ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Animals ; Macrophages/cytology* ; Disease Progression ; Cell Line, Tumor ; Mice ; Amino Acid Transport System ASC/genetics* ; Cell Proliferation ; Lymphatic Metastasis ; Mice, Nude ; Prognosis ; Minor Histocompatibility Antigens

OBJECTIVES: To investigate CEACAM6 expression in nasopharyngeal carcinoma (NPC) and its regulatory effects on tumor cell proliferation, migration, and epithelial-mesenchymal transition (EMT). METHODS: CEACAM6 expression in NPC was analyzed using GEO datasets and validated by immunohistochemistry in NPC tissues and by Western blotting and RT-qPCR in NPC cell lines (HNE1, C666-1, HK1, 5-8F and CNE2Z) and normal nasopharyngeal epithelial NP69 cells. In the NPC cell lines, the effects of lentivirus-mediated CEACAM6 overexpression and knockdown on cell proliferation, migration, invasion and cytoskeletal structures were evaluated using CCK-8 assay, Edu staining, wound healing assay, Transwell assay, and phalloidin staining. Western blotting was performed to determine the expressions of EMT-related proteins (FN1, ITGA5, ITGB1, E-cadherin, N-cadherin and vimentin) in the NPC cells and the effect of FN1 overexpression on ITGA5 and ITGB1 protein expressions. RESULTS: Analysis of the data from the GEO datasets suggested that CEACAM6 was significantly downregulated in NPC, which was associated with poor patient prognosis. Immunohistochemistry also showed low expressions of CEACAM6 in clinical NPC tissues (P<0.05). In NPC cells, CEACAM6 overexpression significantly suppressed cell proliferation, migration and invasion and reduced the fluorescence intensity of actin. CEACAM6 overexpression also resulted in significant downregulation of FN1, ITGA5, ITGB1, N-cadherin and vimentin expressions and upregulation of E-cadherin expression, and FN1 overexpression obviously attenuated the inhibitory effect of CEACAM6 overexpression on ITGA5 and ITGB1 expressions. CONCLUSIONS CEACAM6 inhibits NPC cell migration and invasion by inhibiting EMT via regulating FN1, ITGA5 and ITGB1 expressions.
Humans ; Epithelial-Mesenchymal Transition ; Cell Movement ; Cell Proliferation ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms/metabolism* ; Cell Line, Tumor ; Cell Adhesion Molecules/genetics* ; Antigens, CD/metabolism* ; GPI-Linked Proteins ; Integrin alpha5/metabolism* ; Integrin beta1/metabolism* ; Cadherins/metabolism* ; Fibronectins ; Integrins

OBJECTIVES: To investigate the mechanism by which transforming growth factor‑β (TGF‑β) regulates major histocompatibility complex class I (MHC-I) expression in hepatocellular carcinoma (HCC) cells and its role in immune evasion of HCC. METHODS: HCC cells treated with TGF‑β alone or in combination with SB-431542 (a TGF-β type I receptor inhibitor) were examined for changes in MHC-I expression using RT-qPCR and Western blotting. A RNA interference experiment was used to explore the role of miR-23a-3p/IRF1 signaling in TGF‑β‑mediated regulation of MHC-I. HCC cells with different treatments were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the changes in HCC cell proliferation was assessed using CCK-8 and colony formation assays. T-cell cytotoxicity in the co-culture systems was assessed with lactate dehydrogenase (LDH) release and JC-1 mitochondrial membrane potential assays, and T-cell activation was evaluated by flow cytometric analysis of CD69 cells and ELISA for TNF-α secretion. RESULTS: TGF‑β treatment significantly suppressed MHC-I expression in HCC cells and reduced T-cell activation, leading to increased tumor cell proliferation and decreased HCC cell death in the co-culture systems. Mechanistically, TGF-β upregulated miR-23a-3p, which directly targeted IRF1 to inhibit MHC-I transcription. Overexpression of miR-23a-3p phenocopied TGF‑β‑induced suppression of IRF1 and MHC-I. CONCLUSIONS We reveal a novel immune escape mechanism of HCC, in which TGF‑β attenuates T cell-mediated antitumor immunity by suppressing MHC-I expression through the miR-23a-3p/IRF1 signaling axis.
Humans ; MicroRNAs/genetics* ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Interferon Regulatory Factor-1/metabolism* ; Transforming Growth Factor beta/metabolism* ; Signal Transduction ; Histocompatibility Antigens Class I/metabolism* ; Cell Line, Tumor ; Tumor Escape ; Coculture Techniques

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

Neoantigens exhibit high immunogenic potential and confer a uniqueness to tumor cells, making them ideal targets for personalized cancer immunotherapy. Neoantigens originate from tumor-specific genetic alterations, abnormal viral infections, or other biological mechanisms, including atypical RNA splicing events and post-translational modifications (PTMs). These neoantigens are recognized as foreign by the immune system, eliciting an immune response that largely bypasses conventional mechanisms of central and peripheral tolerance. Advances in next-generation sequencing (NGS), mass spectrometry (MS), and artificial intelligence (AI) have greatly expedited the rapid detection and forecasting of neoantigens, markedly propelling the development of diverse immunotherapeutic strategies, including cancer vaccines, adoptive cell therapy, and antibody treatment. In this review, we comprehensively explore the discovery and characterization of neoantigens and their clinical use within promising immunotherapeutic frameworks. Additionally, we address the current landscape of neoantigen research, the intrinsic challenges of the field, and potential pathways for clinical application in cancer treatment.
Humans ; Neoplasms/therapy* ; Precision Medicine/methods* ; Immunotherapy/methods* ; Antigens, Neoplasm/genetics* ; Cancer Vaccines/immunology* ; High-Throughput Nucleotide Sequencing

The study aimed to investigate the effect of the CD200R1 gene deletion on microglia activation and nigrostriatal dopamine neuron loss in the Parkinson's disease (PD) process. The CRISPR-Cas9 technology was applied to construct the CD200R1^-/- mice. The primary microglia cells of wild-type and CD200R1^-/- mice were cultured and treated with bacterial lipopolysaccharide (LPS). Microglia phagocytosis level was assessed by a fluorescent microsphere phagocytosis assay. PD mouse model was prepared by nigral stereotaxic injection of recombinant adeno-associated virus vector carrying human α-synuclein (α-syn). The changes in the motor behavior of the mice with both genotypes were evaluated by cylinder test, open field test, and rotarod test. Immunohistochemical staining was used to assess the loss of dopamine neurons in substantia nigra. Immunofluorescence staining was used to detect the expression level of CD68 (a key molecule involved in phagocytosis) in microglia. The results showed that CD200R1 deletion markedly enhanced LPS-induced phagocytosis in vitro by the microglial cells. In the mouse model of PD, CD200R1 deletion exacerbated motor behavior impairment and dopamine neuron loss in substantia nigra. Fluorescence intensity analysis results revealed a significant increase in CD68 expression in microglia located in the substantia nigra of CD200R1^-/- mice. The above results suggest that CD200R1 deletion may further activates microglia by promoting microglial phagocytosis, leading to increased loss of the nigrostriatal dopamine neurons in the PD model mice. Therefore, targeting CD200R1 could potentially serve as a novel therapeutic target for the treatment of early-stage PD.
Animals ; Microglia/physiology* ; Mice ; Phagocytosis ; Parkinson Disease/genetics* ; Disease Models, Animal ; Receptors, Cell Surface/physiology* ; Dopaminergic Neurons/pathology* ; Antigens, CD/metabolism* ; Gene Deletion ; Substantia Nigra ; Mice, Inbred C57BL ; Mice, Knockout ; Cells, Cultured ; Male ; alpha-Synuclein ; CD68 Molecule ; Orexin Receptors

Objective This study aims to investigate and analyze the distribution of MN blood type among ethnic minorities in China. Methods Through a systematic retrieval of the 981 literature related to MN blood group distribution, 120 literature, meeting the criteria of this study, with complete data were selected. The literature covers 49 ethnic minorities. SPSS 26 statistical software was used to analyze the data. Results The results showed that among the 49 ethnic minorities in China, the phenotype distribution of MN blood type was MN>MM>NN, with proportions of 42.54%, 41.86%, and 15.06% respectively. The gene frequency for MN blood type exhibited a trend of m>n, with a gene frequency of m being 0.6313 and n being 0.3687. Cluster analysis divided the Chinese ethnic minorities into three groups based on the gene frequency for m, showing the characteristics of Group I>Group II>Group III. Conclusion The MN blood type characteristics in Chinese ethnic minorities show a higher frequency of the M gene compared to the N gene. The frequency of the M gene is higher in southern ethnic minorities than in northern ones. There are significant differences between southwestern ethnic minorities and the Han nationality, but no differences with long-term mixed/settled Han populations.
Humans ; China/ethnology* ; Minority Groups ; Ethnicity/genetics* ; Gene Frequency ; Asian People/genetics* ; Blood Group Antigens/genetics*

Objective To prepare monoclonal antibodies against bovine CD4 and identify their basic biological characteristics. Methods Recombinant bovine CD4 (rHis-BoCD4 and rGST-BoCD4) was successfully expressed and purified by constructing a prokaryotic plasmid of bovine CD4 gene. The bovine CD4 monoclonal antibody was produced using hybridoma technology. The subtype and potency of the monoclonal antibody were identified and analyzed by ELISA, while specificity was analyzed through indirect immunofluorescence assay (IFA) and Western-blot. Results Four hybridoma cell lines, namely, 1H4, 6A10, 3F9 and 4G10, stably secreting monoclonal antibodies against BoCD4 were successfully obtained. The subclasses of the monoclonal antibodies subclass 6A10 was IgG2b and the rest of the monoclonal antibodies were of IgM type. Western-blot results showed that the four anti-bovine CD4 mAb strains were able to specifically bind to the bovine CD4 protein expressed in vitro. Indirect immunofluorescence assay showed that four monoclonal antibodies were able to specifically recognize the natural bovine CD4 protein. Flow cytometry assay showed that 3F9 was best to recognize bovine natural CD4 molecules. Conclusion Four monoclonal antibody strains with high specificity to natural bovine CD4 protein were successfully prepared, which lays the foundation for the subsequent studies on the function of bovine CD4 and diagnosis and treatment of bovine T-lymphocyte diseases.
Animals ; Antibodies, Monoclonal/isolation & purification* ; Cattle ; CD4 Antigens/genetics* ; Hybridomas/immunology* ; Antibody Specificity/immunology* ; Mice ; Mice, Inbred BALB C ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect

Objective To generate and characterize natural killer cell (NK cell)-conditional Cd226 gene knockout mice using Cre-loxP technology, and to explore the role of CD226 on NK cells in alleviating intestinal inflammation in a murine model of ulcerative colitis (UC). Methods NK cell-conditional Cd226 gene knockout mice were generated by crossing loxP-flanked Cd226 mice with Ncr1-Cre mice via the Cre-loxP system. Polymerase chain reaction (PCR) and agarose gel electrophoresis were used for genotyping. A UC model was established by dextran sulfate sodium (DSS) induction. Flow cytometry was performed to analyze CD226 expression levels on NK cells and the infiltration of related immune cells in colon tissues. Hematoxylin-eosin (HE) staining was performed to assess the degree of colonic inflammation. Results DNA gel electrophoresis and flow cytometry confirmed the successful generation of NK cell-specific Cd226 knockout mice. After conditional knockout of Cd226 in NK cells, inflammation in the UC mouse model was alleviated. Flow cytometry results showed a reduced proportion of NK cells in peripheral blood and the colon lamina propria, while HE staining demonstrated attenuated inflammatory responses. Conclusion Specific knockout of Cd226 in NK cells mitigates intestinal inflammation in UC mice by reducing NK cell numbers and inhibiting their pro-inflammatory functions.
Animals ; Colitis, Ulcerative/pathology* ; Killer Cells, Natural/metabolism* ; Mice, Knockout ; T Lineage-Specific Activation Antigen 1 ; Antigens, Differentiation, T-Lymphocyte/genetics* ; Mice ; Disease Models, Animal ; Mice, Inbred C57BL ; Male

Objective To prepare CD318-specific monoclonal antibodies and evaluate their specificity, affinity, and application in immunological detection, laying the foundation for the development of CD318-targeted antibody drugs. MethodsCD318 protein was expressed and purified, and was used as an antigen to immunize mice, then mice with higher antiserum titers were screened. We prepared CD318-specific monoclonal antibodies through cell fusion and monoclonal screening, and the specificity, affinity, and application of the obtained monoclonal antibodies in immunological assays were evaluated. Then we constructed a CD318/CD3-targeting bispecific antibody and assessed its impact on T-cell cytotoxicity. Results Thirteen monoclonal antibodies were successfully generated, with the hybridoma clone 13-8-G2 exhibiting the highest titer, strongest specificity, and broadest applicability. The antibody was identified as an IgG1 isotype with a kappa light chain. The variable region of the light chain measured 318 bp, while the heavy chain variable region was 357 bp, yielding an affinity constant of approximately 7.68×10⁹. The specificity of CD318 was confirmed using flow cytometry and immunofluorescence assays. Additionally, a CD318/CD3-targeting bispecific antibody was constructed using the variable regions of this CD318 monoclonal antibody, which demonstrated enhanced T-cell cytotoxicity. Conclusion High-affinity and highly specific CD318 monoclonal antibodies were successfully prepared, laying a foundation for the development of therapeutic antibodies targeting CD318.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice ; Antibodies, Bispecific/immunology* ; Humans ; Mice, Inbred BALB C ; Antibody Specificity/immunology* ; CD3 Complex/immunology* ; Antigens, CD/genetics* ; T-Lymphocytes/immunology* ; Hybridomas/immunology* ; Female