OBJECTIVE: To analyze the correlation between the expression levels of Tim-3, C-myc and the proportion of T lymphocyte subsets and prognosis in patients with acute lymphoblastic leukemia (ALL). METHODS: The research group selected 60 ALL patients admitted to our hospital from December 2019 to December 2021, while the control group selected 55 healthy volunteers who underwent physical examination in our hospital. The expression levels of Tim-3, C-myc mRNA and the proportion of T lymphocyte subsets in the two groups were detected. The mortality rate of ALL patients was calculated, and the correlation between the expression levels of Tim-3, C-myc, and the proportion of T lymphocyte subsets and pathological features and prognosis was analyzed. RESULTS: Compared with the control group, the levels of Tim-3, C-myc and CD8⁺ in the research group were increased, while the levels of CD3⁺ , CD4⁺ and CD4⁺ /CD8⁺ were decreased (all P < 0.001). The levels of Tim-3, C-myc mRNA, CD3⁺ , CD4⁺ , CD8⁺ , CD4⁺ /CD8⁺ were correlated with risk classification and extramedullary infiltration (all P < 0.05). The survival rate of patients with low expression of Tim-3, C-myc, and CD8⁺ was higher than that of patients with high expression, while the survival rate of patients with high expression of CD3⁺ , CD4⁺ , and CD4⁺ /CD8⁺ was higher than that of patients with low expression (all P < 0.05). Univariate analysis showed that the deceased patients had higher proportions of extramedullary infiltration and high-risk classification, as well as higher levels of Tim-3, C-myc, and CD8⁺ , while lower levels of CD3⁺ , CD4⁺ , and CD4⁺ /CD8⁺ compared with surviving patients (all P < 0.01). Multivariate logistic regression analysis showed that extramedullary invasion, risk classification, Tim-3, C-myc, CD3⁺ , CD4⁺ , CD8⁺ , CD4⁺ /CD8⁺ were the main factors affecting the prognosis of ALL patients (all P < 0.05). ROC curve analysis showed that the combination of Tim-3, C-myc, and T lymphocyte subsets had higher sensitivity and accuracy in predicting prognosis of ALL patients compared with the single diagnosis of Tim-3, C-myc, CD3⁺ , CD4⁺ , CD8⁺ , and CD4⁺ /CD8⁺ (P < 0.05). CONCLUSION ALL patients show higher levels of Tim-3, C-myc mRNA and CD8⁺ but lower levels of CD3⁺ , CD4⁺ and CD4⁺/CD8⁺. Moreover, the expression levels of Tim-3, C-myc, CD3⁺ , CD4⁺ , CD8⁺ and CD4⁺/CD8⁺ are correlated with extramedullary invasion, high-risk classification and prognosis.
Humans ; Hepatitis A Virus Cellular Receptor 2/metabolism* ; Prognosis ; Proto-Oncogene Proteins c-myc/metabolism* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis* ; T-Lymphocyte Subsets ; Male ; Female ; Adult ; Middle Aged ; Adolescent ; RNA, Messenger

OBJECTIVE: To explore the expression and clinical significance of programmed death receptor 1 (PD-1), Th1, Th2, and Th17 cytokines in multiple myeloma (MM). METHODS: A total of 76 MM patients treated in the Tengzhou Central People's Hospital from May 2021 to May 2023 were collected as MM group, and 48 healthy individuals who underwent physical examination during the same period were included as control group. The expression of PD-1 on the surface of CD4⁺ and CD8⁺ T cells and the levels of serum Th1 cytokines ［interleukin (IL) -2, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α)］, Th2 cytokines (IL-4, IL-6, IL-10) and Th17 cytokines (IL-17) were detected in the two groups. Spearman correlation was used to examine the relationship between PD-1, Th1, Th2 and Th17 cytokines and clinical stage and immune typing of MM patients. Multivariate logistic regression analysis was used to analyze the related factors affecting the efficacy of chemotherapy in MM patients, and the factors were tested for multicollinearity. Receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of PD-1, Th1, Th2 and Th17 cytokines in chemotherapy efficacy of MM patients. RESULTS: The levels of CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 in the MM group were higher than those in the control group, while the levels of IL-2, IFN-γ, and TNF-α were lower (all P <0.001). The levels of CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 in R-ISS stage III patients were higher than those in stage II and I patients, and the levels in stage II patients were higher than those in stage I patients (all P <0.05). The IL-2 level in R-ISS stage III patients was lower than that in stage II and I patients, and IL-2 level in R-ISS stage II patients was lower than that in stage I patients (all P <0.05). The levels of CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 in IgG patients were higher than those in IgA, light chain, and non secretory patients, while the level of IL-2 was lower (all P <0.05). Correlation analysis showed that CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 were positively correlated with R-ISS staging in MM patients (r =0.623, 0.635, 0.728, 0.330, 0.742, 0.412), and negatively correlated with immune classification (r =-0.664, -0.756, -0.642, -0.479, -0.613, -0.323). IL-2 was negatively correlated with R-ISS staging in MM patients (r =-0.280), and positively correlated with immune classification (r =0.483). The levels of CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10, and IL-17 in the non-remission group were higher than those in the remission group, while the level of IL-2 was lower (all P <0.001). Multivariate logistic regression analysis showed that the increased CD4⁺T PD-1, CD8⁺T PD-1, IL-4, IL-6, IL-10 and IL-17 were risk factors for the efficacy of chemotherapy in MM patients (OR >1, P <0.05), while the increased IL-2 was a protective factor (OR < 1, P <0.05). The results of multicollinearity test showed that the tolerance of the seven factors included was between 0.714-0.885, and the variance inflation factor was between 1.130-1.400. There was no multicollinearity. The ROC curve analysis results showed that the area under the curve for the combined prediction of chemotherapy efficacy in MM patients by the above 7 factors was 0.942, with specificity of 0.741 and sensitivity of 0.909. CONCLUSION The expression levels of PD-1 on the surface of CD4⁺ and CD8⁺ T cells and serum Th2 and Th17 cytokines in MM patients are high, while Th1 cytokines are low. PD-1, Th1, Th2, and Th17 cytokines are related to clinical stage and immune classification of MM patients. The combined detection of these indicators can help predict the chemotherapy efficacy of MM patients.
Humans ; Programmed Cell Death 1 Receptor/metabolism* ; Multiple Myeloma/blood* ; Cytokines/metabolism* ; Th17 Cells/metabolism* ; Th1 Cells/metabolism* ; Th2 Cells/metabolism* ; Female ; Male ; Interleukin-10 ; Interferon-gamma ; Middle Aged ; Interleukin-17 ; Interleukin-2 ; Interleukin-4 ; Tumor Necrosis Factor-alpha ; Interleukin-6 ; Aged

OBJECTIVE: To analyze the Epstein-Barr virus (EBV) load in different lymphocyte subsets, as well as clinical characteristics and outcomes in patients with hematologic malignancies experiencing EBV reactivation. METHODS: Peripheral blood samples from patients were collected. B, T, and NK cells were isolated sorting with magnetic beads by flow cytometry. The EBV load in each subset was quantitated by real-time quantitative polymerase chain reaction (RT-qPCR). Clinical data were colleted from electronic medical records. Survival status was followed up through outpatient visits and telephone calls. Statistical analyses were performed using SPSS 25.0. RESULTS: A total of 39 patients with hematologic malignancies were included, among whom 35 patients had undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). The median time to EBV reactivation was 4.8 months (range: 1.7-57.1 months) after allo-HSCT. EBV was detected in B, T, and NK cells in 20 patients, in B and T cells in 11 patients, and only in B cells in 4 patients. In the 35 patients, the median EBV load in B cells was 2.19×10⁴ copies/ml, significantly higher than that in T cells (4.00×10³ copies/ml, P <0.01) and NK cells (2.85×10² copies/ml, P <0.01). Rituximab (RTX) was administered for 32 patients, resulting in EBV negativity in 32 patients with a median time of 8 days (range: 2-39 days). Post-treatment analysis of 13 patients showed EBV were all negative in B, T, and NK cells. In the four non-transplant patients, the median time to EBV reactivation was 35 days (range: 1-328 days) after diagnosis of the primary disease. EBV was detected in one or two subsets of B, T, or NK cells, but not simultaneously in all three subsets. These patients received a combination chemotherapy targeting at the primary disease, with 3 patients achieving EBV negativity, and the median time to be negative was 40 days (range: 13-75 days). CONCLUSION In hematologic malignancy patients after allo-HSCT, EBV reactivation commonly involves B, T, and NK cells, with a significantly higher viral load in B cells compared to T and NK cells. Rituximab is effective for EBV clearance. In non-transplant patients, EBV reactivation is restricted to one or two lymphocyte subsets, and clearance is slower, highlighting the need for prompt anti-tumor therapy.
Humans ; Hematologic Neoplasms/virology* ; Herpesvirus 4, Human/physiology* ; Epstein-Barr Virus Infections ; Hematopoietic Stem Cell Transplantation ; Virus Activation ; Lymphocyte Subsets/virology* ; Flow Cytometry ; Killer Cells, Natural/virology* ; Male ; Female ; B-Lymphocytes/virology* ; Viral Load ; Adult ; T-Lymphocytes/virology* ; Middle Aged

OBJECTIVE: To investigate the molecular alterations of splenocytes associated with anti-factor Ⅷ (FⅧ) immune response and the underlying mechanisms based on hemophilia A (HA) murine model via RNA sequencing (RNA-seq) technology. METHODS: Severe HA mice were immunized with recombinant human factor Ⅷ (rhF8) weekly for 4 weeks to establish an FⅧ inhibitor model. High quality raw data were obtained by using bulk RNA-seq and CASAVA base identification technology, and the differentially expressed genes (DEGs) were identified. The DEGs were statistically classified by gene ontology (GO) annotation to obtain information on the major signaling pathways and biological processes involved in anti-FⅧ immune response in HA mouse splenocytes. The cell clusters, genes, and signaling pathway datasets were comprehensively analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and single cell RNA-seq (ScRNA-seq) analysis, respectively. Flow cytometry analysis was used to verify the changes in T follicular helper cells (Tfh) and regulatory T cells (Treg). RESULTS: A total of 3731 DEGs was identified, including 2275 genes with up-regulated expression and 1456 genes with down-regulated expression. The DEGs were enriched in helper T cell differentiation, cytokine receptor, T cell receptor signaling pathway, ferroptosis, etc. Uniform Manifold Approximation and Project (UMAP) downscaling and visualization analysis yielded a total number of 11 T/NK cell subsets, visualizing the overall expression distribution of C-X-C chemokine-specific receptor gene cxcr5 among these T/NK cell subsets. Higher expression of cxcr5 was found in activated Tfh from FⅧ inhibitor mice, in comparison to the control group. The visualization using Upset plot R language showed a close interaction between Tfh and Treg. Moreover, the increased frequencies of Tfh and the decreased frequencies of Treg in inhibitor mouse splenocytes were further verified by flow cytometry analysis. CONCLUSION Multiple immune cell subsets, signaling pathways, and characteristic genes may be involved in the process of anti-FⅧ immune response in HA mouse splenocytes. The molecules involved in the regulation of Tfh/Treg may play key roles, which provide potential biological targets and therapeutic strategies for HA patients with inhibitors in the future.
Animals ; Hemophilia A/genetics* ; Mice ; Sequence Analysis, RNA ; Disease Models, Animal ; Spleen/cytology* ; T-Lymphocytes, Regulatory/immunology* ; Humans ; Signal Transduction ; Factor VIII/immunology* ; T-Lymphocytes, Helper-Inducer/immunology*

Immune thrombocytopenia (ITP) is a hemorrhagic autoimmune disease characterized by antibody-mediated platelet injury. ITP has complicated immunopathological mechanisms that need further elucidation. It is well known that the costimulatory molecules OX40 ligand (OX40L) and OX40 play essential roles in the immunological mechanisms of autoimmune diseases. Previously, we discovered that the expression of OX40L and OX40 is significantly increased in the peripheral blood mononuclear cells (PBMCs) of ITP patients. In our present study, OX40L-induced follicular helper T (Tfh) cells exhibited an activated phenotype with elevated expression of inducible T-cell costimulator (ICOS), programmed cell death protein-1 (PD-1), and cluster of differentiation 40 ligand (CD40L) in vitro. Moreover, aberrant OX40L‒OX40 expression might promote the Tfh1-to-Tfh2 shift in vivo, inducing the generation of autoantibodies by enhancing the helper function of Tfh cells for B lymphocytes in a mouse model, which might accelerate the progression of ITP. Additionally, signal transduction through the OX40L‒OX40 axis might be related to the activation of tumor necrosis factor receptor-associated factor (TRAF)‒nuclear factor-κB (NF-κB) and Janus kinase (JAK)‒signal transducer and activator of transcription (STAT) signaling pathways. Overall, OX40L‒OX40 signaling is proposed as a potential novel therapeutic target for ITP.
Animals ; OX40 Ligand/physiology* ; Purpura, Thrombocytopenic, Idiopathic/immunology* ; Cell Differentiation ; Mice ; T-Lymphocytes, Helper-Inducer/cytology* ; T Follicular Helper Cells/cytology* ; Signal Transduction ; Receptors, OX40 ; Mice, Inbred C57BL ; Humans ; Female

OBJECTIVES: To investigate the therapeutic effects of cimifugin on Crohn's disease (CD)-like colitis in mice and its possible mechanism. METHODS: Thirty adult male C57BL/6 mice were randomized equally into control group, 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis model group, and cimifugin treatment (daily gavage at 12.5 mg/kg) group. The therapeutic effect of cimifugin was evaluated by observing changes in body weight, disease activity index (DAI) scores, colon length, histopathological inflammation scores, and inflammatory cytokine levels in the colonic mucosa. Intestinal barrier integrity in the mice was assessed using immunofluorescence assay and Western blotting for claudin-1 and ZO-1; T-helper (Th) cell subset ratios in the mesenteric lymph nodes were analyzed with flow cytometry. Network pharmacology, KEGG enrichment analysis and molecular docking were used to predict the targets of cimifugin and analyze the key pathways and cimifugin-MAPK protein interactions, which were validated by Western blotting in the mouse models. RESULTS: In mice with TNBS-induced colitis, cimifugin treatment significantly attenuated body weight loss and colon shortening, lowered DAI and histopathological scores, decreased IFN-γ and IL-17 levels, and increased IL-4 and IL-10 levels in the colonic mucosa. Cimifugin treatment also significantly improved TNBS-induced claudin-1 dislocation and reduction of goblet cells, upregulated claudin-1 and ZO-1 expressions, reduced Th1 and Th17 cell percentages, and increased Th2 and Treg cell percentages in the colonic mucosa of the mice. KEGG analysis suggested a possible connection between the effect of cimifugin and MAPK signaling, and molecular docking showed strong binding affinity between cimifugin and MAPK core proteins. Western blotting demonstrated significantly decreased phosphorylation levels of JNK, ERK, and p38 in the colonic mucosa of cimifugin-treated mouse models. CONCLUSIONS Cimifugin alleviates TNBS-induced CD-like colitis by repairing intestinal barrier damage and restoring Th1/Th2 and Th17/Treg balance via suppressing MAPK pathway activation.
Animals ; Mice, Inbred C57BL ; Male ; Mice ; Crohn Disease/immunology* ; Colitis/immunology* ; MAP Kinase Signaling System/drug effects* ; Trinitrobenzenesulfonic Acid ; T-Lymphocytes, Helper-Inducer/drug effects* ; Intestinal Mucosa ; Disease Models, Animal

OBJECTIVES: To investigate the inhibitory effect of pirfenidone (PFD) on growth of bladder cancer xenograft and its regulatory effect on Treg cells in tumor-bearing mice. METHODS: Thirty-two C57BL/6 mice bearing ectopic bladder tumors were randomized into control and PFD groups (n=16). In PFD group, PFD was administered orally at the daily dose of 500 mg/kg, and tumor growth and survival of the mice were monitored. After treatment for 21 days, the tumors and vital organs were harvested for analysis. Immunohistochemistry was used to assess CD3, CD4, CD8, and FOXP3 expressions in the tumors. Flow cytometry and RT-qPCR were used to analyze the percentage of CD4⁺CD25⁺FOXP3⁺ Treg cells and IL-2, IL-10, and IL-35 expressions in the tumors and spleens; organ damage of the mice was examined with HE staining. RESULTS: Compared with the control group, the PFD-treated mice exhibited significantly lower tumor growth rate with smaller tumor volumes at day 21, along with improved survival at day 28. Immunohistochemistry revealed no significant differences in the infiltration of CD3⁺ and CD8⁺ cells between the two groups, but the percentages of CD4⁺ and FOXP3⁺ cells were significantly lower in the tumors of PFD-treated mice. Flow cytometric analysis confirmed a decrease in CD4⁺CD25⁺FOXP3⁺ Treg cells in the tumors from PFD-treated mice, which also had reduced expression levels of IL-2, IL-10 and IL-35 mRNAs in the tumors. No significant differences were found in Treg cell populations or cytokine expressions in the spleen tissues between the two groups. HE staining showed obvious organ damage in neither of the groups. CONCLUSIONS PFD inhibits bladder cancer growth and enhances survival of tumor-bearing mice possibly by suppressing Treg cells in the tumor microenvironment.
Animals ; Urinary Bladder Neoplasms/drug therapy* ; Mice ; T-Lymphocytes, Regulatory/metabolism* ; Mice, Inbred C57BL ; Interleukins/metabolism* ; Interleukin-10/metabolism* ; Cell Line, Tumor ; Interleukin-2/metabolism* ; Xenograft Model Antitumor Assays ; Female

OBJECTIVES: To investigate the therapeutic mechanism of 2,6-dimethoxy-1,4-benzoquinone (DMQ) for alleviating dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. METHODS: Eighteen male C57BL/6J mice were equally randomized into control group, DSS group and DMQ treatment group. In DSS and DMQ groups, the mice were treated with DSS in drinking water to induce UC, and received intraperitoneal injections of sterile PBS or DMQ (20 mg/kg) during modeling. The changes in body weight, disease activity index (DAI), colon length, spleen weight, and colon histological scores of the mice were examined, and the percentages of Th17 and IFN-γ⁺ CD8⁺ T cells in the mesenteric lymph nodes and spleen were analyzed using flow cytometry. The expressions of tight junction proteins (Occludin and ZO-1), proteins associated with inflammasome activation (caspase-1 and p20), IL-1β and TNF-α in the colon tissues were detected using Western blotting or ELISA. In the cell experiment, mouse bone marrow-derived macrophages (BMDMs) primed with lipopolysaccharide (LPS) were treated with DMQ, followed by stmulation with nigericin to activate the classical NLRP3 inflammasome pathway. In cultured human peripheral blood mononuclear cells (PBMCs) treated with either LPS alone or LPS plus nigericin, the effects of DMQ on inflammasome activation, pyroptosis, and cytokine release were evaluated via Western blotting, ELISA, and flow cytometry. RESULTS: In DSS-treated mice, DMQ treatment significantly alleviated DSS-induced body weight loss, colon shortening, spleen enlargement, and colon inflammation. The DMQ-treated mice showed significantly reduced percentages of Th17 cells and IFN-γ⁺ CD8⁺ T cells in the mesenteric lymph nodes and spleen, with increased occludin and ZO-1 expressions and decreased caspase-1 expression in the colon tissue. DMQ obviously inhibited classical NLRP3 inflammasome activation in mouse BMDMs and both the classical and alternative pathways of NLRP3 activation in human PBMCs, causing also suppression of caspase-1-dependent pyroptosis. CONCLUSIONS DMQ ameliorates DSS-induced UC in mice by inhibiting NLRP3 inflammasome activation.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Mice, Inbred C57BL ; Colitis, Ulcerative/metabolism* ; Dextran Sulfate/adverse effects* ; Male ; Inflammasomes/metabolism* ; Mice ; Benzoquinones/therapeutic use* ; Th17 Cells ; Caspase 1/metabolism*

This study investigates the role of Interleukin 17 (IL-17) in exacerbating periapical lesions caused by Porphyromonas gingivalis (Pg) lipopolysaccharides (LPS) in the context of metabolic disease and its potential impact on glucose tolerance. Researchers developed a unique mouse model where mice were monocolonized with Pg to induce periapical lesions. After 1 month, they were fed a high-fat diet (HFD) for 2 months to simulate metabolic disease and oral microbiota dysbiosis. To explore the role of LPS from Pg, wild-type (WT) mice were challenged with purified LPS from Porphyromonas gingivalis, as well as with LPS-depleted and non-depleted Pg bacteria; IL-17 knockout (KO) mice were also included to assess the role of IL-17 signaling. The impact on bone lysis, periapical injury, glucose intolerance, and immune response was assessed. Results showed that in WT mice, the presence of LPS significantly worsened bone lysis, Th17 cell recruitment, and periapical injury. IL-17 KO mice exhibited reduced bone loss, glucose intolerance, and immune cell infiltration. Additionally, inflammatory markers in adipose tissue were lower in IL-17 KO mice, despite increased dysbiosis. The findings suggest that IL-17 plays a critical role in amplifying Pg-induced periapical lesions and systemic metabolic disturbances. Targeting IL-17 recruitment could offer a novel approach to improving glycemic control and reducing type 2 diabetes (T2D) risk in individuals with periapical disease.
Animals ; Porphyromonas gingivalis/immunology* ; Th17 Cells/immunology* ; Lipopolysaccharides/immunology* ; Mice ; Glucose Intolerance/microbiology* ; Interleukin-17/metabolism* ; Mice, Knockout ; Mice, Inbred C57BL ; Disease Models, Animal ; Diet, High-Fat ; Periapical Diseases/microbiology* ; Male ; Dysbiosis

Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic