RESUMO
B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.
Assuntos
Subpopulações de Linfócitos B , Citometria de Fluxo , Imunofenotipagem , Humanos , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Biomarcadores , Fenótipo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Análise Custo-BenefícioRESUMO
Background: Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation. Methods: Liver tissue samples were collected during 2008-2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53). Results: CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells ("hot" cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression. Conclusions: These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with "hot" TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn "cold" into "hot" TIME in ICC.
The tumor immune microenvironment (TIME) plays a critical role in the immune response In many cancers, including intrahepatic cholangiocarcinoma (ICC). Molecular subtyping of the ICC microenvironment already revealed inter-tumoral heterogeneity with variant profiles of immune cell infiltrates. A recent study created an in-depth immune cell atlas of the TIME in biliary tract cancers and could demonstrate the relevance of specific immune cell subpopulations on patient outcome. We are able to provide a distinctive characterization of TIME, separating tumor epithelial- and stroma areas, in a large and representative ICC cohort using digitalized image analysis on tissue microarrays (TMA) as well as multiplex imaging mass cytometry (IMC). The study was designed for identification of immune cell prognosticators allocating institutional ICC patients into a discovery (200815) and a validation (201019) cohort. Immune cell subpopulations were correlated with clinicopathological characteristics and patient outcome. Our results highlight: i. The important role of CD4+ T cell infiltration in ICC patients; ii. ICC tumors with high density of immune cells associated with PD-L1 expression identifies a subset of patients with variant tumor biology; iii. Stimulation of STAT1 pathway may be a relevant target to turn "cold" into "hot" tumors.
Assuntos
Antígeno B7-H1 , Neoplasias dos Ductos Biliares , Biomarcadores Tumorais , Colangiocarcinoma , Microambiente Tumoral , Humanos , Colangiocarcinoma/imunologia , Colangiocarcinoma/patologia , Microambiente Tumoral/imunologia , Masculino , Feminino , Neoplasias dos Ductos Biliares/imunologia , Neoplasias dos Ductos Biliares/patologia , Pessoa de Meia-Idade , Prognóstico , Idoso , Biomarcadores Tumorais/metabolismo , Antígeno B7-H1/metabolismo , Fator de Transcrição STAT1/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Antígenos CD/metabolismo , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Molécula CD68RESUMO
OBJECTIVES: Carcinoembryonic-antigen-related cell-adhesion molecule 1 (CEACAM1) is an adhesion molecule that acts as a coinhibitory receptor in the immune system. We previously demonstrated that CEACAM1 is predominantly expressed on peripheral blood neutrophils in patients with RA. The aim of the present study was to investigate the effects of Janus kinase inhibitors (JAKi) on cytokine-activated human neutrophils and CEACAM1 expression. METHODS: Peripheral blood neutrophils were obtained from healthy subjects. Isolated neutrophils were stimulated with tumor necrosis factor-alpha (TNF-α) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence or absence of JAKi. The expression of CEACAM1 in peripheral blood neutrophils was analyzed by flow cytometry. Protein phosphorylation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 was assessed by western blot using phospho-specific antibodies. RESULTS: We found that TNF-α-induced CEACAM1 expression was marginally suppressed after pretreatment with pan-JAK inhibitor, tofacitinib. Moreover, TNF-α induced STAT1 and STAT3 phosphorylation at the late stimulation phase (4 to 16 h). The expressions of CEACAM1 on neutrophils were markedly up-regulated by GM-CSF not by interleukin (IL)-6 stimulation. All JAKi inhibited GM-CSF-induced CEACAM1 expressions on neutrophils, however, the inhibitory effects of baricitinib were larger compared to those of tofacitinib or filgotinib. Moreover, CEACAM1 was marginally upregulated in interferon (IFN)-γ stimulated neutrophils. Similarly, JAKi inhibited IFN-γ-induced CEACAM1 expressions on neutrophils. CONCLUSIONS: We demonstrated that JAKi prevent GM-CSF-induced CEACAM1 expression in neutrophils, and JAKi-induced inhibition depends on their selectivity against JAK isoforms. These findings suggest that JAKi can modulate the expression of CEACAM1 in cytokine-activated neutrophils, thereby limiting their activation.
Assuntos
Antígenos CD , Moléculas de Adesão Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Inibidores de Janus Quinases , Neutrófilos , Pirimidinas , Fator de Necrose Tumoral alfa , Humanos , Neutrófilos/metabolismo , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Antígenos CD/metabolismo , Pirimidinas/farmacologia , Inibidores de Janus Quinases/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fosforilação/efeitos dos fármacos , Piperidinas/farmacologia , Pirróis/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Citocinas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: The study evaluated the prognostic impact of the immune microenvironment in LSCC with markers of major immune cells to identify the key determinants of short-term disease-free survival (ST DFS) and reveal factors related to disease progression. METHODS: The study cohort included 61 patients who underwent total laryngectomy, 83.6% of whom were male with a mean age of 64.3 years at the time of surgery. Twenty-five patients had long term DFS (over 5 years), 8 - had moderate DFS (between 2 and 5 years), and 28 had short-term DFS (less than 2 years). Immunohistochemical staining and evaluation were performed on samples collected after the laryngectomy. RESULTS: The samples' assessment revealed that the mean expression of all analysed markers was the highest both in stroma and the tumor compartment for short term DFS (ST DFS) patients. Analysis confirmed that a high stromal density of CD8 cells (p = 0.038) significantly correlated with DFS, and that the increased presence of CD57 cells (p = 0.021) was significantly associated with ST DFS. Moreover, the high density of CD68 cells in the tumor epithelial compartment had a negative prognostic impact on DFS (p = 0.032). Analysis of overall survival in the studied cohort with Kaplan-Meyer curves revealed that a high stromal density of CD68 cells was a significant negative predictor of OS (p = 0.008). CONCLUSIONS: The observed associations of CD68 cells infiltration with progression and prognosis in patients with LSCC provide potential screening and therapeutic opportunities for patients with unfavourable outcomes.
Assuntos
Neoplasias Laríngeas , Microambiente Tumoral , Humanos , Microambiente Tumoral/imunologia , Masculino , Neoplasias Laríngeas/patologia , Neoplasias Laríngeas/imunologia , Neoplasias Laríngeas/mortalidade , Neoplasias Laríngeas/cirurgia , Pessoa de Meia-Idade , Feminino , Idoso , Prognóstico , Laringectomia , Intervalo Livre de Doença , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Imunomodulação , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Estadiamento de NeoplasiasRESUMO
BACKGROUND: Sepsis-induced pulmonary injury (SPI) is a common complication of sepsis with a high rate of mortality. N4-acetylcytidine (ac4C) is mediated by the ac4C "writer", N-acetyltransferase (NAT)10, to regulate the stabilization of mRNA. This study aimed to investigate the role of NAT10 in SPI and the underlying mechanism. METHODS: Twenty-three acute respiratory distress syndrome (ARDS) patients and 27 non-ARDS volunteers were recruited. A sepsis rat model was established. Reverse transcription-quantitative polymerase chain reaction was used to detect the expression of NAT10 and transferrin receptor (TFRC). Cell viability was detected by cell counting kit-8. The levels of Fe2+, glutathione, and malondialdehyde were assessed by commercial kits. Lipid reactive oxygen species production was measured by flow cytometric analysis. Western blot was used to detect ferroptosis-related protein levels. Haematoxylin & eosin staining was performed to observe the pulmonary pathological symptoms. RESULTS: The results showed that NAT10 was increased in ARDS patients and lipopolysaccharide-treated human lung microvascular endothelial cell line-5a (HULEC-5a) cells. NAT10 inhibition increased cell viability and decreased ferroptosis in HULEC-5a cells. TFRC was a downstream regulatory target of NAT10-mediated ac4C acetylation. Overexpression of TFRC decreased cell viability and promoted ferroptosis. In in vivo study, NAT10 inhibition alleviated SPI. CONCLUSION: NAT10-mediated ac4C acetylation of TFRC aggravated SPI through promoting ferroptosis.
Assuntos
Ferroptose , Receptores da Transferrina , Sepse , Sepse/metabolismo , Sepse/complicações , Sepse/etiologia , Acetilação , Animais , Humanos , Ratos , Masculino , Receptores da Transferrina/metabolismo , Receptores da Transferrina/genética , Feminino , Lesão Pulmonar/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/patologia , Modelos Animais de Doenças , Acetiltransferases/metabolismo , Acetiltransferases/genética , Pessoa de Meia-Idade , Antígenos CD/metabolismo , Antígenos CD/genética , Citidina/análogos & derivados , Citidina/farmacologia , Linhagem Celular , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/patologia , Ratos Sprague-Dawley , Sobrevivência CelularRESUMO
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a deadly pulmonary disease with impaired immunological response that causes significant tissue damage and organ failure. Postmortem examination of the lung is a useful tool for understanding the immunopathogenesis of this virus. Lung autopsy samples from seven dead SARS-CoV-2 patients were obtained and evaluated using hematoxylin and eosin stain to analyze the histopathological changes in those samples, on the other hand, Immunohistochemical (IHC) staining was used for detection of CD21, CD1a, CR1 (CD35), CD68, Myeloperoxidase (MPO), CD15, CD56, CD3, CD20, CD4, and CD8 cells markers. Histopathological examination revealed diffuse alveolar damage with extensive parenchymal architecture distortion, intravascular fibrin clot, deposition of collagen fibers, vascular congestions and blood vessels containing thrombi, pneumocyte type II with inflammatory cell infiltration. The IHC staining for the innate immune cells such as antigen-presenting cells (APCs) including dendritic cells, Macrophages, and neutrophils showed a strong positive staining, while CD56 Natural killer (NK) cells showed negative staining. On the other hand, the specific immune cells including; CD20 B cells, CD3 T cells, and CD4 helper T cells, showed positive staining while CD8 Cytotoxic T cells showed negative staining. The lung autopsy samples from patients with COVID-19 confirmed the presence of APCs through the positive staining of CD21, CD1a, CD35, CD68, MPO, and CD15 expressed the virus recognition, proinflammatory cytokine production, and adaptive immune cells activation through CD3, CD4, and CD20 positive staining and the role of APCs in the severity of pulmonary infection and pathogenesis of SARS-CoV-2 infection however the absence of the CD56 NK and CD8 cytotoxic T explains the worse infection status for the patients.
Assuntos
Células Apresentadoras de Antígenos , Autopsia , COVID-19 , Pulmão , SARS-CoV-2 , Humanos , COVID-19/imunologia , COVID-19/patologia , Pulmão/patologia , Pulmão/imunologia , Pulmão/virologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , SARS-CoV-2/imunologia , Masculino , Pessoa de Meia-Idade , Feminino , Idoso , Imuno-Histoquímica , Adulto , Antígenos CD/metabolismoRESUMO
OBJECTIVE: To understand the CD8+ tumour infiltrating lymphocyte (TIL) compartment of oesophageal adenocarcinoma (OAC) with regards to markers of lymphocyte exhaustion, tissue residency and to identify possible reasons behind differential responses to therapy. DESIGN: Tumour samples from 44 patients undergoing curative resection for OAC were assessed by flow cytometry for presence of antigen-experienced TILs and markers of activation and exhaustion. Populations of PD-1 and CD39 positive OAC TILs were sorted, and bulk RNA sequencing undertaken using a modified SmartSeq2 protocol. Flow cytometric assessment of functionality was completed. RESULTS: A higher proportion of antigen experienced CD8+ OAC TILs was associated with improved survival following surgery; while, high double positivity (DP) for PD-1 and CD39 among these TILs also correlated significantly with outcome. These DP TILs possess a minority population which is positive for the markers of exhaustion TIM3 and LAG3. Transcriptomic assessment of the PD-1 and CD39 DP TILs demonstrated enrichment for a tissue resident memory T lymphocyte (TRM) phenotype associated with improved survival in other cancers, reinforced by positivity for the canonical TRM marker CD103 by flow cytometry. This population demonstrated maintained functional capacity both in their transcriptomic profile, and on flow cytometric assessment, as well as preserved proliferative capacity. CONCLUSION: Resected OAC are variably infiltrated by PD-1 and CD39 DP TILs, an abundance of which among lymphocytes is associated with improved survival. This DP population has an increased, but still modest, frequency of TIM3 and LAG3 positivity compared to DN, and is in keeping with a functionally competent TRM phenotype.
Assuntos
Adenocarcinoma , Antígenos CD , Apirase , Linfócitos T CD8-Positivos , Neoplasias Esofágicas , Linfócitos do Interstício Tumoral , Receptor de Morte Celular Programada 1 , Humanos , Neoplasias Esofágicas/imunologia , Neoplasias Esofágicas/cirurgia , Neoplasias Esofágicas/patologia , Receptor de Morte Celular Programada 1/metabolismo , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Apirase/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Masculino , Feminino , Antígenos CD/metabolismo , Pessoa de Meia-Idade , Idoso , Prognóstico , Biomarcadores Tumorais , Cadeias alfa de Integrinas/metabolismoRESUMO
Rationale: Understanding the immune mechanisms associated with liver transplantation (LT), particularly the involvement of tissue-resident memory T cells (TRMs), represents a significant challenge. Methods: This study employs a multi-omics approach to analyse liver transplant samples from both human (n = 17) and mouse (n = 16), utilizing single-cell RNA sequencing, bulk RNA sequencing, and immunological techniques. Results: Our findings reveal a comprehensive T cell-centric landscape in LT across human and mouse species, involving 235,116 cells. Notably, we found a substantial increase in CD8+ TRMs within rejected grafts compared to stable ones. The elevated presence of CD8+ TRMs is characterised by a distinct expression profile, featuring upregulation of tissue-residency markers (CD69, CXCR6, CD49A and CD103+/-,), immune checkpoints (PD1, CTLA4, and TIGIT), cytotoxic markers (GZMB and IFNG) and proliferative markers (PCNA and TOP2A) during rejection. Furthermore, there is a high expression of transcription factors such as EOMES and RUNX3. Functional assays and analyses of cellular communication underscore the active role of CD8+ TRMs in interacting with other tissue-resident cells, particularly Kupffer cells, especially during rejection episodes. Conclusions: These insights into the distinctive activation and interaction patterns of CD8+ TRMs suggest their potential utility as biomarkers for graft rejection, paving the way for novel therapeutic strategies aimed at enhancing graft tolerance and improving overall transplant outcomes.
Assuntos
Linfócitos T CD8-Positivos , Rejeição de Enxerto , Transplante de Fígado , Células T de Memória , Análise de Célula Única , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Humanos , Rejeição de Enxerto/imunologia , Animais , Camundongos , Células T de Memória/imunologia , Células T de Memória/metabolismo , Análise de Célula Única/métodos , Análise de Sequência de RNA/métodos , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Memória Imunológica , Masculino , Camundongos Endogâmicos C57BL , Antígenos CD/metabolismo , Antígenos CD/genética , Feminino , Pessoa de Meia-Idade , Proteínas com Domínio TRESUMO
Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103- counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function.
Assuntos
Antígenos CD , Diferenciação Celular , Fatores de Transcrição Forkhead , Cadeias alfa de Integrinas , Linfócitos T Reguladores , Humanos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Antígenos CD/metabolismo , Antígenos CD/imunologia , Cadeias alfa de Integrinas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/imunologia , Fenótipo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Tolerância Imunológica , Receptores Imunológicos/metabolismo , Proteômica/métodos , Receptores CXCR3/metabolismo , Proteína do Gene 3 de Ativação de Linfócitos , Células CultivadasRESUMO
Homologous animal cell product was obtained in protocol developed for female BALB/c mice. Dendritic cell (DC) migration from the injection site into the draining lymph nodes was evaluated. The number of DC labeled with carboxyfluorescein succinimidyl ester (CFSE) in draining lymph nodes increased from 5.3% (16 h) to 13.3% (48 h) (p=0.028) with a maximum at 72 h (15.4%, p=0.003). The immunophenotype of CFSE-DC detected in murine lymph nodes corresponded to the immunophenotype of mature vaccine DCs: they expressed differentiation markers CD11c, CD80, CD83, and CD86 (p>0.05 vs initial DC).
Assuntos
Vacinas Anticâncer , Células Dendríticas , Linfonodos , Camundongos Endogâmicos BALB C , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Vacinas Anticâncer/imunologia , Camundongos , Linfonodos/imunologia , Linfonodos/metabolismo , Succinimidas , Antígenos CD/imunologia , Antígenos CD/metabolismo , Fluoresceínas , Antígeno CD11c/metabolismo , Antígeno CD11c/imunologia , Antígeno B7-2/metabolismo , Antígeno B7-2/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-1/imunologia , Antígeno CD83 , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Diferenciação Celular , Distribuição Tecidual , Imunofenotipagem , Movimento CelularRESUMO
The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood-brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood-brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.
Assuntos
Antígenos CD , Barreira Hematoencefálica , Adesão Celular , Células Endoteliais , Linfócitos T , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/imunologia , Animais , Antígenos CD/metabolismo , Antígenos CD/genética , Células Endoteliais/metabolismo , Células Endoteliais/imunologia , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Camundongos Endogâmicos C57BL , Humanos , Encéfalo/metabolismo , Encéfalo/imunologiaRESUMO
BACKGROUND AND OBJECTIVE: To explore the association between protein quantitative trait loci (pQTL-SNPs) and the risk of LUAD. METHODS: "Blood +" high depth blood proteomics analysis was performed on plasma from female LUAD patients and female healthy controls, and combined with proteomics data from tumors and adjacent non-tumor tissues of female LUAD patients to screen proteins uniformly expressed in plasma and tissues. pQTL-SNPs were then screened through multiple databases and subjected to multilevel screening. The associations between selected pQTL-SNPs and LUAD risk were evaluated by Female Lung Cancer Consortium in Asia GWAS (FLCCA GWAS). Enzyme linked immunosorbent assay (ELISA) is used to determine the levels of candidate protein. RESULTS: A total of 7 pQTL-SNPs were significantly associated with altered LUAD risk (p < 0.05). Meanwhile, the expression of their corresponding target proteins were all decreased in both plasma and tumor tissues of LUAD cases, which may play a role of tumor suppressor proteins. After mutation of 3 pQTL-SNPs (rs7683000, rs73224660, and rs2776937), the expression of corresponding target proteins BST1 and NRP1 decreased, and as potential tumor suppressor proteins, which may promote tumorigenesis and further increasing the risk of developing LUAD (OR >1, p < 0.05); while after mutation the other pQTL-SNP rs62069916, the corresponding target protein APOH expression was increased, while as a potential tumor suppressor protein, which may inhibit tumorigenesis and further reduced the risk of developing LUAD (OR <1, p < 0.05). In addition, the expression of NRP1 and APOH were significant decreased in LUAD cell lines and validated in plasma of LUAD patients. CONCLUSION: A total of 4 pQTL-SNPs (rs7683000, rs73224660, rs2776937, and rs62069916) may associate with altered LUAD risk by regulating the expression of target proteins (BST1, NRP1, and APOH) after mutation.
Assuntos
Adenocarcinoma de Pulmão , Predisposição Genética para Doença , Neoplasias Pulmonares , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Humanos , Feminino , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/sangue , Estudo de Associação Genômica Ampla , Pessoa de Meia-Idade , Proteômica/métodos , Estudos de Casos e Controles , Biomarcadores Tumorais/genética , Proteínas Ligadas por GPI/genética , Antígenos CD/genética , Antígenos CD/metabolismo , IdosoRESUMO
The emergence of lysosome-targeting chimeras (LYTACs), which represents a promising strategy for membrane protein degradation based on lysosomal pathways, has attracted much attention in disease intervention and treatment. However, the expression level of commonly used lysosome-targeting receptors (LTRs) varies in different cell lines, thus limiting the broad applications of LYTACs. To overcome this difficulty, we herein report the development of integrin α3ß1 (ITGA3B1)-facilitated bispecific aptamer chimeras (ITGBACs) as a platform for the degradation of membrane proteins. ITGBACs consist of two aptamers, one targeting ITGA3B1 and another binding to the membrane-associated protein of interest (POI), effectively transporting the POI into lysosomes for degradation. Our findings demonstrate that ITGBACs effectively eliminate pathological membrane proteins, such as CD71 and PTK7, inducing significant cell-cycle arrest and apoptosis and markedly inhibiting tumor growth in tumor-bearing mice models. Therefore, this work provides a novel and versatile membrane protein degradation platform, offering a promising targeted therapy based on tumor-specific LTRs.
Assuntos
Aptâmeros de Nucleotídeos , Receptores da Transferrina , Humanos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Animais , Camundongos , Receptores da Transferrina/metabolismo , Proteínas de Membrana/metabolismo , Proteólise/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/química , Integrina alfa3beta1/metabolismo , Linhagem Celular Tumoral , Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Receptores Proteína Tirosina QuinasesRESUMO
The adhesion receptor vascular endothelial (VE)-cadherin transduces an array of signals that modulate crucial lymphatic cell behaviors including permeability and cytoskeletal remodeling. Consequently, VE-cadherin must interact with a multitude of intracellular proteins to exert these functions. Yet, the full protein interactome of VE-cadherin in endothelial cells remains a mystery. Here, we use proximity proteomics to illuminate how the VE-cadherin interactome changes during junctional reorganization from dis-continuous to continuous junctions, triggered by the lymphangiogenic factor adrenomedullin. These analyses identified interactors that reveal roles for ADP ribosylation factor 6 (ARF6) and the exocyst complex in VE-cadherin trafficking and recycling. We also identify a requisite role for VE-cadherin in the in vitro and in vivo control of secretion of reelin-a lymphangiocrine glycoprotein with recently appreciated roles in governing heart development and injury repair. This VE-cadherin protein interactome shines light on mechanisms that control adherens junction remodeling and secretion from lymphatic endothelial cells.
Assuntos
Junções Aderentes , Antígenos CD , Caderinas , Células Endoteliais , Proteína Reelina , Animais , Humanos , Camundongos , Junções Aderentes/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Caderinas/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Junções Intercelulares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Proteômica/métodos , Serina Endopeptidases/metabolismoRESUMO
Observational studies indicate that variations in peripheral blood mononuclear cell (PBMC) subsets are associated with an increased risk of pulmonary tuberculosis (PTB) and coronavirus disease 2019 (COVID-19), but causal validation is lacking. Here, we combined single-cell expression quantitative trait locus (sc-eQTL) and two-sample mendelian randomization (MR) analyses to elucidate the causal relationship between PBMC subsets and the occurrence of PTB and COVID-19 and verified by RT-qPCR. We observed an increase in the CD4+ Effective Memory T Cell (CD4+ TEM) cluster in both PTB and COVID-19 patients according to the single-cell transcriptional landscape of PBMC. Through MR analysis using an inverse variance weighted (IVW) method, we found strong evidence of positive correlations between CD4+ TEM cell markers (GBP2, TRAV1-2, and ODF2L) and PTB, and between markers (LAG3 and SLFN5) and COVID-19, especially highlighted by lead eQTL-SNPs of GBP2 (rs2256752, p = 4.76321 × 10-15) and LAG3 (rs67706382, p = 6.16× 10-16). Similar results were observed in validation sets, and no pleiotropy was detected in sensitivity analyses including weighted median (WM), MR-Egger, MR-pleiotropy residual sum and outlier, and leave-one-out analyses (all p > 0.05). We visualized the colocalization of marker-eQTLs and markers of PTB and COVID-19 genome-wide association study (GWAS) associations. Based on CellChat analyses, monocytes communicated predominantly with CD4+ TEM cells positively expressing PTB markers (GBP2, TRAV1-2, and ODF2L) and COVID-19 markers (LAG3 and SLFN5) in both PTB and COVID-19. Our data suggest a causal effect between two key CD4+ TEM cell markers (GBP2 and LAG3) and the risk for PTB and COVID-19 infection. Our findings provide novel insights into the biological mechanism for PTB and COVID-19 infection, but future single-cell studies are necessary to further enhance understanding of this find.
Assuntos
Antígenos CD , Linfócitos T CD4-Positivos , COVID-19 , Proteína do Gene 3 de Ativação de Linfócitos , Análise da Randomização Mendeliana , Locos de Características Quantitativas , SARS-CoV-2 , Humanos , COVID-19/genética , COVID-19/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Fatores de Risco , Antígenos CD/genética , Antígenos CD/metabolismo , Análise de Célula Única/métodos , Proteínas de Ligação ao GTP/genética , Células T de Memória/imunologia , Células T de Memória/metabolismo , Biomarcadores , Polimorfismo de Nucleotídeo Único , Masculino , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica AmplaRESUMO
Histiocytoses are rare diseases characterised by infiltration of affected organs by myeloid cells with a monocyte or dendritic cell phenotype. Symptoms can range from self-resolving localised forms to multisystemic lesions requiring specific treatment. To demonstrate extremely rare cases of CD68-negative cardiac histiocytosis with expression of SARS-CoV-2 antigen in infiltrate cells. We demonstrated a case of Erdheim-Chester disease in a 67-year-old man with pericardial involvement and positive dynamics with vemurafenib treatment, an autopsy case of xanthogranulomatous myopericarditis in a 63-year-old man, surgical material of xanthogranulomatous constrictive pericarditis in a 57-year-old man, and an autopsy case of xanthogranulomatosis in a 1-month-old girl. In all cases, xanthogranuloma cells expressed CD163, many of them spike protein SARS-CoV-2, while CD68 expression was detected only in single cells. In this article, we demonstrated four cases of extremely rare CD68-negative cardiac xanthogranulomatosis in three adults and one child with expression of the spike protein SARS-CoV-2 in M2 macrophages. This potential indirect association between COVID-19 and the development of histiocytosis in these patients warrants further investigation. To substantiate this hypothesis, more extensive research is needed.
Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica , COVID-19 , Histiocitose , SARS-CoV-2 , Humanos , COVID-19/metabolismo , COVID-19/patologia , COVID-19/complicações , COVID-19/virologia , Masculino , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos CD/metabolismo , Feminino , Pessoa de Meia-Idade , Idoso , SARS-CoV-2/metabolismo , Histiocitose/patologia , Histiocitose/metabolismo , Doença de Erdheim-Chester/metabolismo , Doença de Erdheim-Chester/patologia , Doença de Erdheim-Chester/diagnóstico , Glicoproteína da Espícula de Coronavírus/metabolismo , Lactente , Molécula CD68 , Receptores de Superfície CelularRESUMO
BACKGROUND: Antibodies that target immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein/ligand 1 (PD-1/PD-L1) are approved for treatment of multiple cancer types. Chemotherapy is often administered with immune checkpoint blockade (ICB) therapies that target CTLA-4 and/or PD-(L)1. ICB targeting other immune checkpoints such as lymphocyte activating gene-3 (LAG-3) has the potential to improve antitumor responses when combined with chemotherapy. Response to anti-PD-1 ICB is dependent on progenitor exhausted CD8+ T cells (TPEX) in the tumor, but it is unclear how chemotherapy alters TPEX proportions and phenotype. METHODS: Here we investigated whether sequential chemotherapy altered TPEX frequency and immune checkpoint expression in multiple murine tumor models. RESULTS: Two doses of two different anti-metabolite chemotherapies increased tumor infiltrating CD4+, and CD8+ TPEX expressing LAG-3 in multiple mouse models, which was not restricted to tumor antigen specific CD8+ T cells. To determine if LAG-3+tumor infiltrating lymphocytes (TILs) could be targeted to improve tumor control, we administered anti-LAG-3 and anti-PD-1 ICB after two doses of chemotherapy and found combination therapy generated robust antitumor responses compared with each agent alone. Both anti-LAG-3 and anti-PD-1 ICB with chemotherapy were required for the complete tumor regression observed. CONCLUSIONS: Changes in immune checkpoint expression on TILs during chemotherapy administration informs selection of ICB therapies to combine with.
Assuntos
Antígenos CD , Inibidores de Checkpoint Imunológico , Proteína do Gene 3 de Ativação de Linfócitos , Linfócitos do Interstício Tumoral , Animais , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Antígenos CD/metabolismo , Feminino , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular TumoralRESUMO
This study aimed to determine the effect of ozone on the expression of Bax and Bcl-2 genes in dental pulp cells. Additionally, the programmed cell death protein 1, programmed death-ligand 1, and CD200 antigens were determined in lymphocytes to assess their surface expression. Dental pulp cells were cultured from extracted healthy third molars and characterized as dental pulp stromal cells. Gene expression of Bcl-2 and Bax was analyzed at 0 s, 6 s, and 12 s of ozone exposure using real-time PCR. Lymphocytes from dental pulp were subjected to ozone exposure for 12 s and PD-1, PD-L1, and CD200/CD200R expression was analyzed by flow cytometry. Upon exposure to ozone for 6 s, the Bcl-2 expression decreased significantly to -0.09, and at 12 s, it increased significantly to 0.3. Bax gene expression level increased significantly to 0.188 after 6 s exposure, and at 12 s, to 0.16. Lymphocytes exposed to ozone for 12 s showed minimal changes in PD-1, PD-L1, and CD200/CD200R expression levels, indicating that oxidative stress does not impact the signaling pathways regulating these molecules. The significant upregulation of Bcl-2 at 12 s highlights the cells' effort to protect themselves from prolonged oxidative stress, possibly tipping the balance toward cell survival and tissue repair. However, the absence of changes in PD-1 and PD-L1 expression on lymphocytes under oxidative stress suggests that these molecules are not sensitive to oxidative stress in this context.
Assuntos
Antígenos CD , Apoptose , Antígeno B7-H1 , Polpa Dentária , Ozônio , Receptor de Morte Celular Programada 1 , Humanos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Apoptose/efeitos dos fármacos , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Células Cultivadas , Estresse Oxidativo , Projetos Piloto , Regulação da Expressão Gênica/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/imunologia , Linfócitos/efeitos dos fármacos , Adulto Jovem , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Adulto , Transdução de Sinais/efeitos dos fármacosRESUMO
Background: Autoimmune hepatitis (AIH) is a serious liver disease characterized by immune disorders, particularly effector T-cell overactivation. This study aimed to explore the therapeutic effect and underlying mechanism of mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment on CD4+ T-cell overactivation and liver injury in AIH. Methods: The metabolic changes of CD4+ T cells were assayed in human AIH and mouse hepatitis models. The liver protective effect of MSC-EVs was evaluated by transaminase levels, liver histopathology and inflammation. The effect of MSC-EVs on the metabolic state of CD4+ T cells was also explored. Results: Enhanced glycolysis (eg, ~1.5-fold increase in hexokinase 2 levels) was detected in the CD4+ T cells of AIH patient samples and mouse hepatitis models, whereas the inhibition of glycolysis decreased CD4+ T-cell activation (~1.8-fold decrease in CD69 levels) and AIH liver injury (~6-fold decrease in aminotransferase levels). MSC-EV treatment reduced CD4+ T-cell activation (~1.5-fold decrease in CD69 levels) and cytokine release (~5-fold decrease in IFN-γ levels) by reducing glycolysis (~3-fold decrease) while enhancing mitochondrial oxidative phosphorylation (~2-fold increase in maximal respiration) in such cells. The degree of liver damage in AIH mice was ameliorated after MSC-EV treatment (~5-fold decrease in aminotransferase levels). MSC-EVs carried abundant mitochondrial proteins and might transfer them to metabolically reprogram CD4+ T cells, whereas disrupting mitochondrial transfer impaired the therapeutic potency of MSC-EVs in activated CD4+ T cells. Conclusion: Disordered glucose metabolism promotes CD4+ T-cell activation and associated inflammatory liver injury in AIH models, which can be reversed by MSC-EV therapy, and this effect is at least partially dependent on EV-mediated mitochondrial protein transfer between cells. This study highlights that MSC-EV therapy may represent a new avenue for treating autoimmune diseases such as AIH.
Assuntos
Linfócitos T CD4-Positivos , Vesículas Extracelulares , Hepatite Autoimune , Células-Tronco Mesenquimais , Animais , Hepatite Autoimune/terapia , Hepatite Autoimune/metabolismo , Hepatite Autoimune/imunologia , Linfócitos T CD4-Positivos/metabolismo , Humanos , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Glicólise , Masculino , Proteínas Mitocondriais/metabolismo , Feminino , Modelos Animais de Doenças , Fígado/metabolismo , Ativação Linfocitária , Camundongos Endogâmicos C57BL , Hexoquinase/metabolismo , Mitocôndrias/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD/metabolismo , Fosforilação Oxidativa , Reprogramação Metabólica , Lectinas Tipo CRESUMO
The integrity of the blood-labyrinth barrier (BLB) is essential for inner ear homeostasis, regulating the ionic composition of endolymph and perilymph and preventing harmful substance entry. Endothelial hyperpermeability, central in inflammatory and immune responses, is managed through complex intercellular communication and molecular signaling pathways. Recent studies link BLB permeability dysregulation to auditory pathologies like acoustic trauma, autoimmune inner ear diseases, and presbycusis. Polymorphonuclear granulocytes (PMNs), or neutrophils, significantly modulate vascular permeability, impacting endothelial barrier properties. Neutrophil extracellular traps (NETs) are involved in diseases with autoimmune and autoinflammatory bases. The present study evaluated the impact of NETs on a BLB cellular model using a Transwell® setup. Our findings revealed a concentration-dependent impact of NETs on human inner ear-derived endothelial cells. In particular, endothelial permeability markers increased, as indicated by reduced transepithelial electrical resistance, enhanced dextran permeability, and downregulated junctional gene expression (ZO1, OCL, and CDH5). Changes in cytoskeletal architecture were also observed. These preliminary results pave the way for further research into the potential involvement of NETs in BLB impairment and implications for auditory disorders.