ABSTRACT
CD8+ T cells are essential for adaptive immunity against infection and tumors. Their ability to proliferate after stimulation is crucial to their functionality. Dendritic cells (DCs) are professional antigen-presenting cells that induce their proliferation. Here, we show that thapsigargin-induced LAD2 mast cell (MC) line-released products can impair the ability of monocyte-derived DCs to induce CD8+ T-cell proliferation and the generation of Th1 cytokine-producing T cells. We found that culture medium conditioned with LAD2 MCs previously stimulated with thapsigargin (thapsLAD2) induces maturation of DCs as determined by the maturation markers CD80, CD83, CD86, and HLA-DR. However, thapsLAD2-matured DCs produced no detectable TNFα or IL-12 during the maturation. In addition, although their surface expression of PD-L1 was comparable with the immature or TLR7/8-agonist (R848)-matured DCs, their TIM-3 expression was significantly higher than in immature DCs and even much higher than in R848-matured DCs. In addition, contrary to R848-matured DCs, the thapsLAD2-matured DCs only tended to induce enhanced proliferation of CD4+ T cells than immature DCs. For CD8+ T cells, this tendency was not even detected because thapsLAD2-matured and immature DCs comparably induced their proliferation, which contrasted with the significantly enhanced proliferation induced by R848-matured DCs. Furthermore, these differences were comparably recapitulated in the ability of the tested DCs to induce IFNγ- and IFNγ/TNFα-producing T cells. These findings show a novel mechanism of MC-mediated regulation of adaptive immune responses.
Subject(s)
CD8-Positive T-Lymphocytes , Cell Differentiation , Cell Proliferation , Dendritic Cells , Lymphocyte Activation , Mast Cells , Thapsigargin , Humans , Dendritic Cells/immunology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Thapsigargin/pharmacology , Cell Proliferation/drug effects , Cell Differentiation/drug effects , Mast Cells/immunology , Mast Cells/drug effects , Mast Cells/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Monocytes/immunology , Monocytes/drug effects , Monocytes/metabolism , Hepatitis A Virus Cellular Receptor 2/metabolism , Cytokines/metabolism , Imidazoles/pharmacology , Cell LineABSTRACT
Acute myeloid leukaemia (AML) is a biologically heterogeneous haematological malignancy. This study was performed to identify the potential biomarkers for the prognosis and treatment of AML. We applied weighted gene co-expression network analysis to identify key modules and hub genes related to the prognosis of AML using data from The Cancer Genome Atlas (TCGA). In total, 1581 differentially expressed genes (1096 upregulated and 485 downregulated) were identified between AML patients and healthy controls, with the blue module being the most significant among 14 modules associated with AML morphology. Through functional enrichment analysis, we identified 217 genes in the blue module significantly enriched in 'neutrophil degranulation' and 'neutrophil activation involved in immune response' pathways. The survival analysis revealed six genes (S100A9, S100A8, HK3, CD93, CXCR2 and FGL2) located in the significantly enriched pathway that were notably related to AML survival. We validated the expression of these six genes at gene and single-cell levels and identified methylation loci of each gene, except for S100A8. Finally, in vitro experiments were performed to demonstrate whether the identified hub genes were associated with AML survival. After knockdown of CD93 and FGL2, cell proliferation was significantly reduced in U937 cell line over 5 days. In summary, we identified CD93 and FGL2 as key hub genes related to AML survival, with FGL2 being a novel biomarker for the prognosis and treatment of AML.
Subject(s)
Biomarkers, Tumor , Gene Regulatory Networks , Leukemia, Myeloid, Acute , Receptors, Complement , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/metabolism , Biomarkers, Tumor/genetics , Prognosis , Receptors, Complement/genetics , Receptors, Complement/metabolism , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Gene Expression Regulation, Leukemic , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Hepatitis A Virus Cellular Receptor 2/genetics , Hepatitis A Virus Cellular Receptor 2/metabolism , Gene Expression Profiling , Cell Line, Tumor , DNA Methylation/genetics , Survival Analysis , FibrinogenABSTRACT
Despite the success of immunotherapy, overcoming immunoresistance in cancer remains challenging. We identified a unique niche of tumor-associated macrophages (TAMs), coexpressing T cell immunoglobulin and mucin domain-containing 3 (TIM3) and V-domain immunoglobulin suppressor of T cell activation (VISTA), that dominated human and mouse tumors resistant to most of the currently used immunotherapies. TIM3+VISTA+ TAMs were sustained by IL-4-enriching tumors with low (neo)antigenic and T cell-depleted features. TIM3+VISTA+ TAMs showed an anti-inflammatory and protumorigenic phenotype coupled with inability to sense type I interferon (IFN). This was established with cancer cells succumbing to immunogenic cell death (ICD). Dying cancer cells not only triggered autocrine type I IFNs but also exposed HMGB1/VISTA that engaged TIM3/VISTA on TAMs to suppress paracrine IFN-responses. Accordingly, TIM3/VISTA blockade synergized with paclitaxel, an ICD-inducing chemotherapy, to repolarize TIM3+VISTA+ TAMs to proinflammatory TAMs that killed cancer cells via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. We propose targeting TIM3+VISTA+ TAMs to overcome immunoresistant tumors.
Subject(s)
Hepatitis A Virus Cellular Receptor 2 , Immunotherapy , Tumor-Associated Macrophages , Tumor-Associated Macrophages/metabolism , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/drug effects , Animals , Hepatitis A Virus Cellular Receptor 2/metabolism , Humans , Immunotherapy/methods , Mice , Drug Resistance, Neoplasm , Neoplasms/therapy , Neoplasms/immunology , Neoplasms/drug therapy , Neoplasms/pathology , Cell Line, Tumor , Tumor Microenvironment/immunology , Interferon Type I/metabolism , B7 AntigensABSTRACT
OBJECTIVE: Aim: To clarify the association between response to Trastuzumab and molecular expression of TIM-3 and FOXP-3 immune checkpoints. PATIENTS AND METHODS: Materials and Methods: FOXP-3 and TIM-3 expression in peripheral blood was analyzed using qPCR, and the serum level of Trastuzumab was estimated using an immune sorbent enzyme assay. RESULTS: Results: During treatment with Trastuzumab, the FOXP-3 gene expression showed a significant decline throughout one year of treatment, going from 0.85 at cycle 9 to 0.75 at cycle 17. While the TIM-3 gene expression showed a significant up regulation at cycle 9 to 2.8 fold, followed by a reduction in the fold change from 2.8 to 1.7 in the font of reference gene expression. CONCLUSION: Conclusions:FOXP-3 and TIM-3 have the potential to be suggestive markers that can anticipate the response to Trastuzumab, but they are not capable of predicting the likelihood of recurrence.
Subject(s)
Breast Neoplasms , Receptor, ErbB-2 , Trastuzumab , Humans , Trastuzumab/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Female , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Hepatitis A Virus Cellular Receptor 2/genetics , Hepatitis A Virus Cellular Receptor 2/metabolism , Antineoplastic Agents, Immunological/therapeutic use , Middle Aged , Adult , Biomarkers, Tumor/genetics , Biomarkers, Tumor/blood , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
BACKGROUND: Sinonasal mucosal melanoma (SNMM) is a rare but aggressive tumor with a poor prognosis. The co-inhibitory receptors T cell immunoglobulin and mucinodomain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) are promising new targets in anti-cancer immunotherapy. The expression profiles of these immune checkpoint molecules (ICMs) and potential prognostic implications have not been characterized in SNMM yet. METHODS: Immunohistochemical staining for TIGIT, LAG-3 and TIM-3 was performed on tumor tissue samples from 27 patients with primary SNMM. Associations between ICM expression and demographic parameters, AJCC tumor stage, overall survival, and recurrence-free survival were retrospectively analyzed. RESULTS: SNMM patients with low numbers of TIGIT+ and TIM-3+ tumor infiltrating lymphocytes (TILs) in the primary tumor survived significantly longer than patients with a high degree of TIGIT+ and TIM-3+ TILs. High infiltration with TIM-3+ or TIGIT+ lymphocytes was associated with the higher T4 stage and decreased 5-year survival. CONCLUSION: We identified high densities of TIM-3+ and TIGIT+ TILs as strong negative prognostic biomarkers in SNMM. This suggests that TIM-3 and TIGIT contribute to immunosuppression in SNMM and provides a rationale for novel treatment strategies based on this next generation of immune checkpoint inhibitors. Prospective studies with larger case numbers are warranted to confirm our findings and their implications for immunotherapy.
Subject(s)
Hepatitis A Virus Cellular Receptor 2 , Lymphocytes, Tumor-Infiltrating , Melanoma , Receptors, Immunologic , Humans , Hepatitis A Virus Cellular Receptor 2/metabolism , Male , Receptors, Immunologic/metabolism , Receptors, Immunologic/analysis , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Female , Middle Aged , Melanoma/pathology , Melanoma/immunology , Melanoma/mortality , Melanoma/metabolism , Aged , Prognosis , Adult , Biomarkers, Tumor/analysis , Biomarkers, Tumor/metabolism , Retrospective Studies , Aged, 80 and over , Paranasal Sinus Neoplasms/pathology , Paranasal Sinus Neoplasms/immunology , Paranasal Sinus Neoplasms/metabolism , Paranasal Sinus Neoplasms/mortality , Nasal Mucosa/pathology , Nasal Mucosa/immunology , Nasal Mucosa/metabolismABSTRACT
Preeclampsia is a disorder of pregnancy characterized by endothelial dysfunction, abnormal placentation, systemic inflammation, and altered immune reaction. The aim of this study was to investigate the immune checkpoint molecules TIM-3 and Gal-9 on macrophages and Hofbauer cells (HBC) in the placenta of preeclampsia patients. Immunohistochemistry and Immunofluorescence was used to characterize the expression of the macrophage markers CD68 and CD163, CK7 and the proteins TIM-3 and Gal-9 in the placentas of preeclampsia patients comparing it to the placentas of healthy pregnancies. Double immunofluorescence staining (TIM-3 with CD3/CD19/CD56) was used to analyze the TIM-3 expression on other immune cells (T cells, B cells, NK cells) within the chorionic villi. The expression of TIM-3 on decidual macrophages did not significantly differ between the preeclamptic and the control group (p = 0.487). When looking at the different offspring we saw an upregulation of TIM-3 expression on decidual macrophages in preeclamptic placentas with female offspring (p = 0.049). On Hofbauer cells within the chorionic villi, the TIM-3 expression was significantly downregulated in preeclamptic cases without a sex-specific difference (p < 0.001). Looking at the protein Gal-9 the expression was proven to be downregulated both, on decidual macrophages (p = 0.003) and on Hofbauer cells (p = 0.002) within preeclamptic placentas compared to healthy controls. This was only significant in male offspring (p < 0.001 and p = 0.013) but not in female offspring (p = 0.360 and p = 0.068). While TIM-3 expression within the extravillious trophoblast and the syncytiotrophoblast was significantly downregulated (p < 0.001 and p = 0.012) in preeclampsia, the expression of Gal-9 was upregulated in (p < 0.001 and p < 0.001) compared to healthy controls. The local variations of the immune checkpoint molecules TIM-3 and Gal-9 in the placenta may contribute to the inflammation observed in preeclamptic patients. It could therefore contribute to the pathogenesis and be an important target in the treatment of preeclampsia.
Subject(s)
Galectins , Hepatitis A Virus Cellular Receptor 2 , Macrophages , Placenta , Pre-Eclampsia , Humans , Pregnancy , Hepatitis A Virus Cellular Receptor 2/metabolism , Female , Pre-Eclampsia/immunology , Pre-Eclampsia/metabolism , Pre-Eclampsia/pathology , Macrophages/immunology , Macrophages/metabolism , Galectins/metabolism , Adult , Placenta/immunology , Placenta/metabolism , Placenta/pathology , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Receptors, Cell Surface/metabolism , Chorionic Villi/immunology , Chorionic Villi/metabolism , Chorionic Villi/pathology , MaleABSTRACT
BACKGROUND: Immunotherapy has revolutionized the treatment of various types of tumors, but there has been no breakthrough in the treatment of gliomas. The aim of this study is to discover valuable immunotherapy target in glioma, analyze its expression in glioma and the related microenvironment, explore potential immunotherapy strategies, and propose new possibilities for the treatment of gliomas. METHODS: Immunohistochemistry (IHC) and multiplex fluorescence immunohistochemistry (mIHC) were used to analyze the expression of common immune markers and checkpoints in 187 glioma patients from Sun Yat-sen University Caner Center (SYSUCC). Bioinformatics analysis was used to examine the expression of TIM-3 in different macrophages using the Chinese Glioma Genome Atlas (CGGA) single-cell sequencing database. The Kaplan-Meier curve was used to predict the prognostic value of samples with high TIM-3 and CD68 expression. The R package was used to analyze the somatic mutation status and the sensitivity of small molecule inhibitors in TIM-3/CD68 double-high expression samples. RESULTS: TIM-3 is a relatively highly expressed immune checkpoint in glioma. Unlike other tumors, TIM-3 is mainly expressed on macrophages in the glioma microenvironment. TIM-3/CD68 double-high expression suggests poor survival in glioma and may be a new upgrade marker in both IDH-mutant glioma and IDH-wildtype low-grade glioma (LGG) glioma (P < 0.01). Exploring the combination of TIM-3 inhibitors and p38 MAPK inhibitor may be a potential treatment direction for TIM-3/CD68 double high expression gliomas in the future. CONCLUSIONS: The combination of TIM-3 and CD68 holds significant importance as a potential target for both prognosis and therapeutic intervention in glioma.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , Biomarkers, Tumor , Brain Neoplasms , Glioma , Hepatitis A Virus Cellular Receptor 2 , Tumor Microenvironment , Humans , Hepatitis A Virus Cellular Receptor 2/metabolism , Hepatitis A Virus Cellular Receptor 2/genetics , Glioma/metabolism , Glioma/therapy , Glioma/genetics , Glioma/mortality , Glioma/diagnosis , Brain Neoplasms/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/therapy , Brain Neoplasms/mortality , Prognosis , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, CD/metabolism , Antigens, CD/genetics , Tumor Microenvironment/immunology , Female , Male , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/genetics , Middle Aged , Immunotherapy/methods , Adult , Macrophages/immunology , Macrophages/metabolism , Gene Expression Regulation, Neoplastic , CD68 MoleculeABSTRACT
BACKGROUND: Toxoplasma gondii infection causes adverse pregnancy outcomes by affecting the expression of immunotolerant molecules in decidual immune cells. Galectin-9 (Gal-9) is widely expressed in decidual macrophages (dMφ) and is crucial for maintaining normal pregnancy by interacting with the immunomodulatory protein T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3). However, the effects of T. gondii infection on Gal-9 expression in dMφ, and the impact of altered Gal-9 expression levels on the maternal-fetal tolerance function of decidual natural killer (dNK) cells, are still unknown. METHODS: Pregnancy outcomes of T. gondii-infected C57BL/6 and Lgals9-/- pregnant mice models were recorded. Expression of Gal-9, c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and Forkhead box protein O1 (FOXO1) was detected by western blotting, flow cytometry or immunofluorescence. The binding of FOXO1 to the promoter of Lgals9 was determined by chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR). The expression of extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), cAMP-response element binding protein (CREB), phosphorylated CREB (p-CREB), T-box expressed in T cells (T-bet), interleukin 10 (IL-10), and interferon gamma (IFN-γ) in dNK cells was assayed by western blotting. RESULTS: Toxoplasma gondii infection increased the expression of p-JNK and FOXO1 in dMφ, resulting in a reduction in Gal-9 due to the elevated binding of FOXO1 with Lgals9 promoter. Downregulation of Gal-9 enhanced the phosphorylation of ERK, inhibited the expression of p-CREB and IL-10, and promoted the expression of T-bet and IFN-γ in dNK cells. In the mice model, knockout of Lgals9 aggravated adverse pregnancy outcomes caused by T. gondii infection during pregnancy. CONCLUSIONS: Toxoplasma gondii infection suppressed Gal-9 expression in dMφ by activating the JNK/FOXO1 signaling pathway, and reduction of Gal-9 contributed to dysfunction of dNK via Gal-9/Tim-3 interaction. This study provides new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.
Subject(s)
Galectins , Killer Cells, Natural , Macrophages , Mice, Inbred C57BL , Toxoplasma , Toxoplasmosis , Animals , Female , Pregnancy , Galectins/genetics , Galectins/metabolism , Mice , Killer Cells, Natural/immunology , Macrophages/immunology , Toxoplasma/immunology , Toxoplasmosis/immunology , Decidua/immunology , Mice, Knockout , Hepatitis A Virus Cellular Receptor 2/genetics , Hepatitis A Virus Cellular Receptor 2/metabolism , Pregnancy Outcome , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/metabolismABSTRACT
PROBLEM: Endometriosis is a prevalent chronic gynecological disease linked to immune dysfunction. The protein T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) plays a crucial role in immune system balance. Malfunction of TIM-3 may result in excessive immune activation and inflammatory tissue damage. Given TIM-3's established role in the development of cancer and autoimmune diseases, we decided to study its role in women suffering from endometriosis. STUDY METHOD: We included a total of 62 female patients, all of whom had undergone laparoscopic surgery. Of these, 47 had endometriosis and 15 did not. During surgery, we collected peritoneal fluid (PF) and peripheral blood (PB) samples from every patient for analysis using flow cytometry. To mark the samples, we used a panel of monoclonal antibodies and examined TIM-3 expression in their immune cells. RESULTS: Endometriosis patients in PB demonstrated a significantly lower percentage of CD56+ T cells with TIM-3 expression. As endometriosis progressed through its stages, this expression lessened. This decrease was particularly notable in women with stage III/IV endometriosis. Additionally, both women diagnosed with intestinal endometriosis and those with recent endometriosis diagnoses showed a significantly reduced percentage of CD56+ T cells expressing TIM-3. CONCLUSIONS: Patients with endometriosis exhibit diminished TIM-3 expression within circulating T cells. This warrants further investigation to discern whether it contributes to the progression of endometriosis, potentially through the amplification of autoreactive T cells and inflammation.
Subject(s)
Endometriosis , Hepatitis A Virus Cellular Receptor 2 , Adult , Female , Humans , Middle Aged , Ascitic Fluid/immunology , Ascitic Fluid/metabolism , Endometriosis/immunology , Endometriosis/metabolism , Hepatitis A Virus Cellular Receptor 2/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Young AdultABSTRACT
Introduction: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Methods: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. Results: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. Conclusion: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.
Subject(s)
Hepatitis A Virus Cellular Receptor 2 , Immunotherapy, Adoptive , Mesothelin , Receptors, Chimeric Antigen , Uterine Cervical Neoplasms , Xenograft Model Antitumor Assays , Humans , Animals , Hepatitis A Virus Cellular Receptor 2/metabolism , Hepatitis A Virus Cellular Receptor 2/genetics , Female , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/therapy , Uterine Cervical Neoplasms/genetics , Mice , Immunotherapy, Adoptive/methods , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Cell Line, Tumor , Tumor Microenvironment/immunology , Mice, SCIDABSTRACT
Relapse and treatment resistance pose significant challenges in the management of pediatric B cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). The efficacy of immunotherapy in leukemia remains limited due to factors such as the immunosuppressive tumor microenvironment (TME) and lack of suitable immunotherapeutic targets. Thus, an in-depth characterization of the TME in pediatric leukemia is warranted to improve the efficacy of immunotherapy. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the TME of pediatric B-ALL and AML, focusing specifically on bone-marrow-derived T cells. Moreover, we investigated the transcriptome changes during the initiation, remission, and relapse stages of pediatric AML. Our findings revealed that specific functional expression programs correlated with fluctuations in various T cell subsets, which may be associated with AML progression and relapse. Furthermore, our analysis of cellular communication networks led to the identification of VISTA, CD244, and TIM3 as potential immunotherapeutic targets in pediatric AML. Finally, we detected elevated proportions of γδ T cells and associated functional genes in samples from pediatric patients diagnosed with B-ALL and AML, which could inform the development of novel therapeutic approaches, potentially focusing on γδ T cells.
Subject(s)
Leukemia, Myeloid, Acute , Single-Cell Analysis , Tumor Microenvironment , Humans , Tumor Microenvironment/immunology , Tumor Microenvironment/genetics , Single-Cell Analysis/methods , Child , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Transcriptome , Hepatitis A Virus Cellular Receptor 2/genetics , Hepatitis A Virus Cellular Receptor 2/metabolism , Gene Expression Profiling/methods , Child, Preschool , Male , Female , B7 Antigens/genetics , Adolescent , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Gene Expression Regulation, LeukemicABSTRACT
Plasma gelsolin (pGSN) overexpression in ovarian cancer (OVCA) disarms immune function, contributing to chemoresistance. The aim of this study was to investigate the immunoregulatory effects of pGSN expression on natural killer (NK) cell function in OVCA. OVCA tissues from primary surgeries underwent immunofluorescent staining of pGSN and the activated NK cell marker natural cytotoxicity triggering receptor 1 to analyze the prognostic impact of pGSN expression and activated NK cell infiltration. The immunoregulatory effects of pGSN on NK cells were assessed using apoptosis assay, cytokine secretion, immune checkpoint-receptor expression, and phosphorylation of STAT3. In OVCA tissue analyses, activated NK cell infiltration provided survival advantages to patients. However, high pGSN expression attenuated the survival benefits of activated NK cell infiltration. In the in vitro experiment, pGSN in OVCA cells induced NK cell death through cell-to-cell contact. pGSN increased T-cell immunoglobulin and mucin-domain-containing-3 expression (TIM-3) on activated NK cells. Further, it decreased interferon-γ production in activated TIM-3+ NK cells, attenuating their anti-tumor effects. Thus, increased pGSN expression suppresses the anti-tumor functions of NK cells. The study provides insights into why immunotherapy is rarely effective in patients with OVCA and suggests novel treatment strategies.
Subject(s)
Carcinoma, Ovarian Epithelial , Drug Resistance, Neoplasm , Gelsolin , Killer Cells, Natural , Ovarian Neoplasms , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Killer Cells, Natural/drug effects , Female , Gelsolin/metabolism , Gelsolin/blood , Carcinoma, Ovarian Epithelial/pathology , Carcinoma, Ovarian Epithelial/immunology , Carcinoma, Ovarian Epithelial/drug therapy , Drug Resistance, Neoplasm/drug effects , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Ovarian Neoplasms/drug therapy , Cell Line, Tumor , Middle Aged , Hepatitis A Virus Cellular Receptor 2/metabolism , Apoptosis/drug effects , STAT3 Transcription Factor/metabolism , Interferon-gamma/metabolismABSTRACT
AIM: To investigate the status of immune checkpoint molecules (CTLA-4 and TIM-3) in meningiomas and thus contribute to the development of new personalized treatment strategies. MATERIAL AND METHODS: We utilized 402 cases of meningioma for this study. New blocks were prepared using the tissue microarray method, and sections obtained from these blocks were immunohistochemically stained with CTLA-4 and TIM-3 antibodies. Subsequently, statistical analysis were performed. RESULTS: Our findings revealed that CTLA-4 expression were observed in 25.1% of meningiomas. CTLA-4 expression and the number of expressing lymphocytes were found to be significantly higher in high-grade tumors and in those with brain invasion. Meningiomas with staining of immune cells with TIM-3 are 3.5%, and the tumor grade was correlated with the number of immune cells expressing TIM-3. CONCLUSION: Immune checkpoint molecules (CTLA-4 and TIM-3) with varying levels of expression can serve as prognostic and predictive biomarkers, as well as important targets for therapy. Drugs developed for CTLA-4 and TIM-3 molecules may prove to be more effective in treating meningiomas with high-grade, brain-invading, spontaneous necrosis, and macronucleolus.
Subject(s)
CTLA-4 Antigen , Hepatitis A Virus Cellular Receptor 2 , Immunohistochemistry , Meningeal Neoplasms , Meningioma , Humans , Meningioma/immunology , Meningioma/pathology , Meningioma/metabolism , Male , Meningeal Neoplasms/immunology , Meningeal Neoplasms/pathology , Meningeal Neoplasms/metabolism , Female , Middle Aged , CTLA-4 Antigen/metabolism , CTLA-4 Antigen/immunology , Hepatitis A Virus Cellular Receptor 2/metabolism , Aged , Adult , Biomarkers, Tumor/metabolism , Immune Checkpoint Proteins/metabolism , Aged, 80 and over , Young Adult , AdolescentABSTRACT
BACKGROUND: There is growing evidence indicating immune inflammation is a key factor in the progression of chronic obstructive pulmonary disease (COPD). Immune checkpoints (ICs) are crucial targets for modulating the functional activation and differentiation of immune cells, particularly in relation to immune inflammation and the regulation of T cell activation and exhaustion. However, the precise mechanisms of ICs in COPD remain understood. METHODS: COPD datasets were obtained from the Gene Expression Omnibus (GEO) and analyzed using GEO2R and Limma to identify differentially expressed genes. LASSO regression was then applied to screen ICs closely associated with COPD. Finally, target genes were selected based on gene expression profiles. Gene ontology (GO), immune infiltration analysis, and gene set enrichment analysis (GSEA) were utilized to assess the relationship between IC genes (ICGs) and immune cells. Subsequently, tobacco-exposed mice, anti-Tim3-treated mice, and HAVCR2-knockout mice were generated, with flow cytometry being used to confirm the results. RESULTS: Through the analysis of GSE38974 and LASSO regression, five ICGs were identified. Subsequent validation using GSE20257 and GSE76925 confirmed these findings. Gene expression profiling highlighted HAVCR2 as having the strongest correlation with COPD. Further investigation through immune infiltration analysis, GO, and GSEA indicated a link between HAVCR2 and CD8+ T cells in COPD. Flow cytometry experiments demonstrated high Tim3 expression in CD8+ T cells of mice exposed to tobacco, promoting Tc1 and inhibiting Tc17, thus affecting CD8+ Tem activation and CD8+ Tcm formation, leading to an immune imbalance within CD8+ T cells. CONCLUSION: Prolonged exposure to tobacco upregulates Tim3 in CD8+ T cells, triggering its regulatory effects on Tc1/Tc17. Knocking out HAVCR2 further upregulated the expression of CD8+ Tem while suppressing the expression of CD8+ Tcm, indicating that Tim3 plays a role in the activation and differentiation of CD8+ T cells in the context of tobacco exposure.
Subject(s)
Computational Biology , Hepatitis A Virus Cellular Receptor 2 , Pulmonary Disease, Chronic Obstructive , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/immunology , Animals , Humans , Mice , Computational Biology/methods , Hepatitis A Virus Cellular Receptor 2/genetics , Hepatitis A Virus Cellular Receptor 2/metabolism , Gene Expression Profiling/methods , Mice, Knockout , Immune Checkpoint Proteins/genetics , Immune Checkpoint Proteins/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Mice, Inbred C57BL , MaleABSTRACT
BACKGROUND: Advancements in immunotherapeutic approaches only had a modest impact on the therapy of lung neuroendocrine neoplasms (LNENs). Our multicenter study aimed to investigate the expression patterns of novel immunotherapy targets in intermediate- and high-grade LNENs. METHODS: The expressions of V-domain Ig suppressor of T cell activation (VISTA), OX40L, Glucocorticoid-induced TNF receptor (GITR), and T cell immunoglobulin and mucin domain 3 (TIM3) proteins were measured by immunohistochemistry in surgically resected tumor samples of 26 atypical carcinoid (AC), 49 large cell neuroendocrine lung cancer (LCNEC), and 66 small cell lung cancer (SCLC) patients. Tumor and immune cells were separately scored. RESULTS: Tumor cell TIM3 expression was the highest in ACs (p < 0.001), whereas elevated tumor cell GITR levels were characteristic for both ACs and SCLCs (p < 0.001 and p = 0.011, respectively). OX40L expression of tumor cells was considerably lower in ACs (vs. SCLCs; p < 0.001). Tumor cell VISTA expression was consistently low in LNENs, with no significant differences across histological subtypes. ACs were the least immunogenic tumors concerning immune cell abundance (p < 0.001). Immune cell VISTA and GITR expressions were also significantly lower in these intermediate-grade malignancies than in SCLCs or in LCNECs. Immune cell TIM3 and GITR expressions were associated with borderline prognostic significance in our multivariate model (p = 0.057 and p = 0.071, respectively). CONCLUSIONS: LNEN subtypes have characteristic and widely divergent VISTA, OX40L, GITR, and TIM3 protein expressions. By shedding light on the different expression patterns of these immunotherapy targets, the current multicenter study provides support for the future implementation of novel immunotherapeutic approaches.
Subject(s)
Biomarkers, Tumor , Glucocorticoid-Induced TNFR-Related Protein , Hepatitis A Virus Cellular Receptor 2 , Immunotherapy , Lung Neoplasms , Neuroendocrine Tumors , Humans , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Lung Neoplasms/metabolism , Male , Female , Hepatitis A Virus Cellular Receptor 2/metabolism , Immunotherapy/methods , Neuroendocrine Tumors/immunology , Neuroendocrine Tumors/metabolism , Neuroendocrine Tumors/therapy , Neuroendocrine Tumors/pathology , Middle Aged , Aged , Glucocorticoid-Induced TNFR-Related Protein/metabolism , Biomarkers, Tumor/metabolism , B7 Antigens/metabolism , Adult , Neoplasm Grading , OX40 Ligand/metabolism , Prognosis , Aged, 80 and overABSTRACT
Objective: The aberrant mobilization and activation of various T lymphocyte subpopulations play a pivotal role in the pathogenesis of diabetic kidney disease (DKD), yet the regulatory mechanisms underlying these processes remain poorly understood. Our study is premised on the hypothesis that the dysregulation of immune checkpoint molecules on T lymphocytes disrupts kidney homeostasis, instigates pathological inflammation, and promotes DKD progression. Methods: A total of 360 adult patients with DKD were recruited for this study. The expression of immune checkpoint molecules on T lymphocytes was assessed by flow cytometry for peripheral blood and immunofluorescence staining for kidney tissue. Single-cell sequencing (scRNA-seq) data from the kidneys of DKD mouse model were analyzed. Results: Patients with DKD exhibited a reduction in the proportion of CD3+TIM-3+ T cells in circulation concurrent with the emergence of significant albuminuria and hematuria (p=0.008 and 0.02, respectively). Conversely, the incidence of infection during DKD progression correlated with an elevation of peripheral CD3+TIM-3+ T cells (p=0.01). Both univariate and multivariate logistic regression analysis revealed a significant inverse relationship between the proportion of peripheral CD3+TIM-3+ T cells and severe interstitial mononuclear infiltration (OR: 0.193, 95%CI: 0.040,0.926, p=0.04). Immunofluorescence assays demonstrated an increase of CD3+, TIM-3+ and CD3+TIM-3+ interstitial mononuclear cells in the kidneys of DKD patients as compared to patients diagnosed with minimal change disease (p=0.03, 0.02 and 0.002, respectively). ScRNA-seq analysis revealed decreased gene expression of TIM3 on T lymphocytes in DKD compared to control. And one of TIM-3's main ligands, Galectin-9 on immune cells showed a decreasing trend in gene expression as kidney damage worsened. Conclusion: Our study underscores the potential protective role of TIM-3 on T lymphocytes in attenuating the progression of DKD and suggests that monitoring circulating CD3+TIM3+ T cells may serve as a viable strategy for identifying DKD patients at heightened risk of disease progression.
Subject(s)
Diabetic Nephropathies , Hepatitis A Virus Cellular Receptor 2 , T-Lymphocytes , Hepatitis A Virus Cellular Receptor 2/metabolism , Humans , Diabetic Nephropathies/immunology , Diabetic Nephropathies/etiology , Diabetic Nephropathies/pathology , Female , Middle Aged , Male , Animals , Mice , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Aged , Adult , Inflammation/immunology , Kidney/pathology , Kidney/immunology , Mice, Inbred C57BL , Disease ProgressionABSTRACT
Acute myeloid leukemia (AML) is an aggressive heterogeneous disease characterized by several alterations of the immune system prompting disease progression and treatment response. The therapies available for AML can affect lymphocyte function, limiting the efficacy of immunotherapy while hindering leukemia-specific immune reactions. Recently, the treatment based on Venetoclax (VEN), a specific B-cell lymphoma 2 (BCL-2) inhibitor, in combination with hypomethylating agents (HMAs) or low-dose cytarabine, has emerged as a promising clinical strategy in AML. To better understand the immunological effect of VEN treatment, we characterized the phenotype and immune checkpoint (IC) receptors' expression on CD4+ and CD8+ T cells from AML patients after the first and second cycle of HMA in combination with VEN. HMA and VEN treatment significantly increased the percentage of naïve CD8+ T cells and TIM-3+ CD4+ and CD8+ T cells and reduced cytokine-secreting non-suppressive T regulatory cells (Tregs). Of note, a comparison between AML patients treated with HMA only and HMA in combination with VEN revealed the specific contribution of VEN in modulating the immune cell repertoire. Indeed, the reduction of cytokine-secreting non-suppressive Tregs, the increased TIM-3 expression on CD8+ T cells, and the reduced co-expression of PD-1 and TIM-3 on both CD4+ and CD8+ T cells are all VEN-specific. Collectively, our study shed light on immune modulation induced by VEN treatment, providing the rationale for a novel therapeutic combination of VEN and IC inhibitors in AML patients.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , CD8-Positive T-Lymphocytes , Hepatitis A Virus Cellular Receptor 2 , Leukemia, Myeloid, Acute , Programmed Cell Death 1 Receptor , Sulfonamides , T-Lymphocytes, Regulatory , Humans , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/drug therapy , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Sulfonamides/administration & dosage , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Hepatitis A Virus Cellular Receptor 2/metabolism , Hepatitis A Virus Cellular Receptor 2/antagonists & inhibitors , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Middle Aged , Aged , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Female , Male , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Adult , Aged, 80 and overABSTRACT
Immune-checkpoint blockade (ICB) reinvigorates T cells from exhaustion and potentiates T-cell responses to tumors. However, most patients do not respond to ICB therapy, and only a limited response can be achieved in a "cold" tumor with few infiltrated lymphocytes. Synthetic biology can be used to engineer bacteria as controllable bioreactors to synthesize biotherapeutics in situ. We engineered attenuated Salmonella VNP20009 with synthetic gene circuits to produce PD-1 and Tim-3 scFv to block immunosuppressive receptors on exhausted T cells to reinvigorate their antitumor response. Secreted PD-1 and Tim-3 scFv bound PD-1+ Tim-3+ T cells through their targeting receptors in vitro and potentiated the T-cell secretion of IFN-γ. Engineered bacteria colonized the hypoxic core of the tumor and synthesized PD-1 and Tim-3 scFv in situ, reviving CD4+ T cells and CD8+ T cells to execute an antitumor response. The bacteria also triggered a strong innate immune response, which stimulated the expansion of IFN-γ+ CD4+ T cells within the tumors to induce direct and indirect antitumor immunity.
Subject(s)
Immune Checkpoint Inhibitors , Programmed Cell Death 1 Receptor , Salmonella , Immune Checkpoint Inhibitors/pharmacology , Animals , Programmed Cell Death 1 Receptor/metabolism , Programmed Cell Death 1 Receptor/immunology , Mice , Salmonella/immunology , Salmonella/genetics , Hepatitis A Virus Cellular Receptor 2/metabolism , Hepatitis A Virus Cellular Receptor 2/genetics , Cell Line, Tumor , CD8-Positive T-Lymphocytes/immunology , Humans , Interferon-gamma/metabolism , Interferon-gamma/immunology , Single-Chain Antibodies/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/pharmacology , Mice, Inbred C57BL , Synthetic Biology/methods , CD4-Positive T-Lymphocytes/immunology , Immunotherapy/methodsABSTRACT
Toll-like receptor 8 (TLR8), an important innate immune receptor recognizing single stranded RNA and the antiviral imidazoquinoline compounds, can activate intracellular signaling pathway and produce an inflammatory response to kill and eliminate pathogens. However, the molecular regulation mechanisms of TLR8 signaling and its anti-infection activity are not fully elucidated. Our previous transcriptome analysis of porcine TLR8 (pTLR8) signaling suggested the immune checkpoint receptor TIM-3 as the potential regulator for pTLR8. Here we investigated TIM-3 in the regulation of pTLR8 signaling and its anti-infection activity. Our results showed that porcine TIM-3 is upregulated by pTLR8 signaling and TIM-3 inhibits pTLR8 signaling activity in a negative feedback way. Accordingly, TIM-3 disturbs pTLR8 mediated anti-bacterial and anti-viral activity. Mechanistically, TIM-3 suppresses PI3K-AKT pathway by inhibiting the TLR8-PI3K p85 interaction and subsequent AKT phosphorylation which is essential for TLR8 signaling and anti-infection activity. Therefore, our study reveals new insights into innate immune TLR8 signaling and its anti-infection function.
Subject(s)
Hepatitis A Virus Cellular Receptor 2 , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , Toll-Like Receptor 8 , Animals , Humans , Hepatitis A Virus Cellular Receptor 2/metabolism , Immunity, Innate/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Swine , Toll-Like Receptor 8/metabolism , HEK293 Cells , Vero CellsABSTRACT
Chronic infections induce CD4+ T-cells with cytotoxic functions (CD4 CTLs); at present, it is still unknown whether latent tuberculosis (LTB) and active tuberculosis (ATB) induce CD4 CTLs. Plasma and cells from four patient groups-uninfected contact (UC), LTB, and ATB (divided as sensitive [DS-TB]- or resistant [DR-TB]-drug)-were evaluated by flow cytometry, q-PCR, and proteomics. The data showed that ATB patients had an increased frequency of CD4+ T-cells and a decreased frequency of CD8+ T-cells. The latter displays an exhausted-like profile characterized by CD39, CD279, and TIM-3 expression. ATB had a high frequency of CD4 + perforin+ cells, suggesting a CD4 CTL profile. The expression (at the transcriptional level) of granzyme A, granzyme B, granulysin, and perforin, as well as the genes T-bet (Tbx21) and NKG2D (Klrk1), in enriched CD4+ T-cells, confirmed the cytotoxic signature of CD4+ T-cells during ATB (which was stronger in DS-TB than in DR-TB). Moreover, proteomic analysis revealed the presence of HSP70 (in DS-TB) and annexin A5 (in DR-TB), which are molecules that have been associated with favoring the CD4 CTL profile. Finally, we found that lipids from Mycobacterium tuberculosis increased the presence of CD4 CTLs in DR-TB patients. Our data suggest that ATB is characterized by exhausted-like CD8+ T-cells, which, together with a specific microenvironment, favor the presence of CD4 CTLs.