RESUMO
Introduction: α-galactosylceramide (α-GalCer), a prototypical agonist of invariant natural killer T (iNKT) cells, stimulates iNKT cells to produce various cytokines such as IFNγ and IL4. Moreover, repeated α-GalCer treatment can cause protective or pathogenic outcomes in various immune-mediated diseases. However, the precise role of α-GalCer-activated iNKT cells in sepsis development remains unclear. To address this issue, we employed a lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced murine sepsis model and two alternative models. Methods: Sepsis was induced in wild-type (WT) C57BL/6 (B6) mice by three methods (LPS/D-GalN, α-GalCer/D-GalN, and cecal slurry), and these mice were monitored for survival rates. WT B6 mice were intraperitoneally injected with α-GalCer or OCH (an IL4-biased α-GalCer analog) one week prior to the induction of sepsis. To investigate the effects of α-GalCer-mediated iNKT cell activation on sepsis development, immune responses were analyzed by flow cytometry using splenocytes and liver-infiltrating leukocytes. In addition, a STAT6 inhibitor (AS1517499) and an IL10 inhibitor (AS101) were employed to evaluate the involvement of IL4 or IL10 signaling. Furthermore, we performed B cell adoptive transfers to examine the contribution of α-GalCer-induced regulatory B (Breg) cell populations in sepsis protection. Results: In vivo α-GalCer pretreatment polarized iNKT cells towards IL4- and IL10-producing phenotypes, significantly attenuating LPS/D-GalN-induced septic lethality in WT B6 mice. Furthermore, α-GalCer pretreatment reduced the infiltration of immune cells to the liver and attenuated pro-inflammatory cytokine production. Treatment with a STAT6 inhibitor was unable to modulate disease progression, indicating that IL4 signaling did not significantly affect iNKT cell-mediated protection against sepsis. This finding was confirmed by pretreatment with OCH, which did not alter sepsis outcomes. However, interestingly, prophylactic effects of α-GalCer on sepsis were significantly suppressed by treatment with an IL10 antagonist, suggesting induction of IL10-dependent anti-inflammatory responses. In addition to IL10-producing iNKT cells, IL10-producing B cell populations were significantly increased after α-GalCer pretreatment. Conclusion: Overall, our results identify α-GalCer-mediated induction of IL10 by iNKT and B cells as a promising option for controlling the pathogenesis of postoperative sepsis.
Assuntos
Galactosilceramidas , Interleucina-10 , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais , Choque Séptico , Animais , Galactosilceramidas/farmacologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Camundongos , Interleucina-10/metabolismo , Choque Séptico/imunologia , Modelos Animais de Doenças , Linfócitos B/imunologia , Linfócitos B/metabolismo , Masculino , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologiaRESUMO
Unlike conventional CD4+ T cells, which are phenotypically and functionally plastic, invariant NKT (iNKT) cells generally exist in a terminally differentiated state. Naïve CD4+ T cells can acquire alternative epigenetic states in response to different cues, but it remains unclear whether peripheral iNKT cells are epigenetically stable or malleable. Repetitive encounters of liver-resident iNKT cells (LiNKTs) with alpha-galactosylceramide (αGalCer)/CD1d-coated nanoparticles (NPs) can trigger their differentiation into a LiNKT cell subset expressing a T regulatory type 1 (TR1)-like (LiNKTR1) transcriptional signature. Here we dissect the epigenetic underpinnings of the LiNKT-LiNKTR1 conversion as compared to those underlying the peptide-major histocompatibility complex (pMHC)-NP-induced T-follicular helper (TFH)-to-TR1 transdifferentiation process. We show that gene upregulation during the LINKT-to-LiNKTR1 cell conversion is associated with demethylation of gene bodies, inter-genic regions, promoters and distal gene regulatory elements, in the absence of major changes in chromatin exposure or deposition of expression-promoting histone marks. In contrast, the naïve CD4+ T cell-to-TFH differentiation process involves extensive remodeling of the chromatin and the acquisition of a broad repertoire of epigenetic modifications that are then largely inherited by TFH cell-derived TR1 cell progeny. These observations indicate that LiNKT cells are epigenetically malleable and particularly susceptible to gene de-methylation.
Assuntos
Metilação de DNA , Epigênese Genética , Fígado , Células T Matadoras Naturais , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Animais , Fígado/imunologia , Fígado/citologia , Fígado/metabolismo , Camundongos , Diferenciação Celular/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Camundongos Endogâmicos C57BL , Galactosilceramidas , Transcrição Gênica , Antígenos CD1d/genética , Antígenos CD1d/metabolismoRESUMO
Due to the genetic diversity between the mother and the fetus, heightened control over the immune system during pregnancy is crucial. Immunological parameters determined by clinicians in women with idiopathic recurrent spontaneous abortion (RSA) include the quantity and activity of Natural Killer (NK) and Natural Killer T (NKT) cells, the quantity of regulatory T lymphocytes, and the ratio of pro-inflammatory cytokines, which indicate imbalances in Th1 and Th2 cell response. The processes are controlled by immune checkpoint proteins (ICPs) expressed on the surface of immune cells. We aim to investigate differences in the expression of ICPs on T cells, T regulatory lymphocytes, NK cells, and NKT cells in peripheral blood samples collected from RSA women, pregnant women, and healthy multiparous women. We aim to discover new insights into the role of ICPs involved in recurrent pregnancy loss. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from blood samples obtained from 10 multiparous women, 20 pregnant women (11-14th week of pregnancy), and 20 RSA women, at maximum of 72 h after miscarriage. The PBMCs were stained for flow cytometry analysis. Standard flow cytometry immunophenotyping of PBMCs was performed using antibodies against classical lymphocyte markers, including CD3, CD4, CD8, CD56, CD25, and CD127. Additionally, ICPs were investigated using antibodies against Programmed Death Protein-1 (PD-1, CD279), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3, CD366), V-domain Ig suppressor of T cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), and Lymphocyte activation gene 3 (LAG-3). We observed differences in the surface expression of ICPs in the analyzed subpopulations of lymphocytes between early pregnancy and RSA, after miscarriage, and in women. We noted diminished expression of PD-1 on T lymphocytes (p = 0.0046), T helper cells (CD3CD4 positive cells, p = 0.0165), T cytotoxic cells (CD3CD8 positive cells, p = 0.0046), T regulatory lymphocytes (CD3CD4CD25CD127 low positive cells, p = 0.0106), and NKT cells (CD3CD56/CD16 positive cells, p = 0.0438), as well as LAG-3 on lymphocytes T (p = 0.0225) T helper, p = 0.0426), T cytotoxic cells (p = 0.0458) and Treg (p = 0.0293), and cells from RSA women. Impaired expression of TIM-3 (p = 0.0226) and VISTA (p = 0.0039) on CD8 cytotoxic T and NK (TIM3 p = 0.0482; VISTA p = 0.0118) cells was shown, with an accompanying increased expression of TIGIT (p = 0.0211) on NKT cells. The changes in the expression of surface immune checkpoints indicate their involvement in the regulation of pregnancy. The data might be utilized to develop specific therapies for RSA women based on the modulation of ICP expression.
Assuntos
Aborto Habitual , Biomarcadores , Proteínas de Checkpoint Imunológico , Células Matadoras Naturais , Humanos , Feminino , Gravidez , Aborto Habitual/imunologia , Aborto Habitual/metabolismo , Aborto Habitual/sangue , Adulto , Biomarcadores/sangue , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteínas de Checkpoint Imunológico/metabolismo , Proteínas de Checkpoint Imunológico/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/imunologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Antígenos CD/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Objective: Acute respiratory distress syndrome (ARDS) presents a global health challenge, characterized by significant morbidity and mortality. However, the role of natural killer T (NKT) cells in human ARDS remains poorly understood. Therefore, this study explored the numerical and functional status of NKT cells in patients with ARDS, examining their clinical relevance and interactions with macrophages and fibroblasts during various stages of the syndrome. Methods: Peripheral blood from 40 ARDS patients and 30 healthy controls was analyzed, with paired samples of peripheral blood and bronchoalveolar lavage fluid (BALF) from seven ARDS patients. We measured levels of NKT cells, cytokines, CD69, programmed death-1 (PD-1), and annexin-V using flow cytometry, and extracellular matrix (ECM) protein expression using real-time PCR. Results: ARDS patients exhibited decreased circulating NKT cells with elevated CD69 expression and enhanced IL-17 production. The reduction in NKT cells correlated with PaO2/FiO2 ratio, albumin, and C-reactive protein levels. Proliferative responses to α-galactosylceramide (α-GalCer) were impaired, and co-culturing NKT cells with monocytes or T cells from ARDS patients resulted in a reduced α-GalCer response. Increased and activated NKT cells in BALF induced proinflammatory cytokine release by macrophages and ECM protein expression in fibroblasts. Conclusion: ARDS is associated with a numerical deficiency but functional activation of circulating NKT cells, showing impaired responses to α-GalCer and altered interactions with immune cells. The increase in NKT cells within BALF suggests their role in inducing inflammation and remodeling/fibrosis, highlighting the potential of targeting NKT cells as a therapeutic approach for ARDS.
Assuntos
Células T Matadoras Naturais , Síndrome do Desconforto Respiratório , Humanos , Síndrome do Desconforto Respiratório/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Adulto , Idoso , Macrófagos/imunologia , Macrófagos/metabolismo , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/imunologia , Ativação Linfocitária/imunologia , Antígenos de Diferenciação de Linfócitos T , Antígenos CD , Lectinas Tipo CRESUMO
BACKGROUND: We aimed to investigate whether alpha-galactosylceramide (α-GalCer)-producing Bacteroides fragilis could induce natural killer T (NKT) cells in nonobese diabetic (NOD) mice and reduce their diabetes incidence. METHODS: Five-week-old female NOD mice were treated orally with B. fragilis, and islet pathology and diabetes onset were monitored. Immune responses were analyzed by flow cytometry and multiplex technology. Effects of ultraviolet (UV)-killed α-GalCer-producing B. fragilis and their culture medium on invariant NKT (iNKT) cells were tested ex vivo on murine splenocytes, and the immunosuppressive capacity of splenocytes from B. fragilis-treated NOD mice were tested by adoptive transfer to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. RESULTS: B. fragilis reduced the diabetes incidence from 69% to 33% and the percent of islets with insulitis from 40% to 7%, which doubled the serum insulin level compared with the vehicle-treated control mice. Furthermore, the early treatment reduced proinflammatory mediators in the serum, whereas the proportion of CD4+ NKT cell population was increased by 33%. B. fragilis growth media stimulated iNKT cells and anti-inflammatory M2 macrophages ex vivo in contrast to UV-killed bacteria, which had no effect, strongly indicating an α-GalCer-mediated effect. Adoptive transfer of splenocytes from B. fragilis-treated NOD mice induced a similar diabetes incidence as splenocytes from untreated NOD mice. CONCLUSIONS: B. fragilis induced iNKT cells and M2 macrophages and reduced type 1 diabetes in NOD mice. The protective effect seemed to be more centered on gut-pancreas interactions rather than a systemic immunosuppression. B. fragilis should be considered for probiotic use in individuals at risk of developing type 1 diabetes.
Assuntos
Bacteroides fragilis , Galactosilceramidas , Camundongos Endogâmicos NOD , Células T Matadoras Naturais , Probióticos , Animais , Feminino , Galactosilceramidas/farmacologia , Probióticos/uso terapêutico , Probióticos/farmacologia , Camundongos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Diabetes Mellitus Tipo 1/prevenção & controle , Diabetes Mellitus Tipo 1/metabolismo , Incidência , Camundongos SCIDRESUMO
BACKGROUND: Granzymes are essential serine proteases in cytotoxic T cells and natural killer (NK) cells, with GZMK's expression being less understood. This study aims to uncover GZMK expression profiles across various immune cell types using single-cell RNA sequencing meta-analysis. OBJECTIVE: This study aims to uncover GZMK expression profiles across various immune cell types using single-cell RNA sequencing meta-analysis. METHODS: We conducted a meta-analysis using cellxgene, an interactive data exploration platform developed by the Chan Zuckerberg Initiative. We focused on mature T cells, NK cells, B cells, and NKT cells. We also checked transcription factor binding sites at the granzyme gene promoter regions using JASPAR. Comparative analysis was also done using mouse single-cell RNA sequencing data. RESULTS: GZMK was the most lowly expressed in NK cells and mature NKT cells in most tissues except for colon and lymph nodes. In mature T cells, GZMK is similarly or more highly expressed than other granzymes. HBCA data revealed weak expression of GZMK in NK cells but strong expression in effector memory CD8-positive, alpha-beta T cells. Combined data shows no significant difference in GZMK expression between cell types. Subtype analysis shows that GZMK expression was higher in CD16-negative, CD56-bright NK cells when compared to CD16-positive, CD56-dim NK cells. We also identified unique transcription factor binding sites for GZMK. While this pattern in mouse data with low Gzmk expression in NK cells and higher T cells was repeated. CONCLUSION: GZMK expression is distinctively regulated among immune cells and tissues, with unique promoter regions and transcription factor binding sites contributing to this differential expression. These insights into GZMK's role in immune function and regulation offer potential therapeutic targets.
Assuntos
Granzimas , Células Matadoras Naturais , Análise de Célula Única , Granzimas/genética , Granzimas/metabolismo , Animais , Análise de Célula Única/métodos , Camundongos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/imunologia , Humanos , RNA-Seq/métodos , Linfócitos B/metabolismo , Linfócitos B/imunologia , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/imunologia , Regiões Promotoras Genéticas , Sítios de Ligação , Análise da Expressão Gênica de Célula ÚnicaRESUMO
To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d-TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR-ceramide-CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the "sphingomyelin cycle" that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.
Assuntos
Antígenos CD1d , Lipidômica , Células T Matadoras Naturais , Receptores de Antígenos de Linfócitos T , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Animais , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Camundongos , Lipidômica/métodos , Humanos , Autoantígenos/imunologia , Autoantígenos/metabolismo , Ceramidas/metabolismo , Ceramidas/imunologia , Lipídeos/química , Lipídeos/imunologia , Estresse do Retículo Endoplasmático/imunologiaRESUMO
Objective: The immune response initiated by SARS-CoV-2 infection in pregnancy is poorly elucidated. We aimed to access and compare the antiviral cellular responses and lymphocytes activation between healthy pregnancies and pregnant women infected with SARS-CoV-2. Methods: We detected the immunological changes of lymphocytes in peripheral blood of healthy non-pregnant women, non-pregnant women with COVID-19, healthy pregnant women, pregnant women with COVID-19 and convalescent group by flow cytometry. In vitro blockade was used to identify NKT-like cell activation through ICOS-ICOSL pathway. Results: We found that CD3+CD56+ NKT-like cells decreased significantly in COVID-19 positive pregnant women compared to healthy pregnant women. NKT-like cells of pregnant women expressed higher level of activating receptors CD69 and NKp46 after SARS-CoV-2 infection. Particularly, they also increased the expression of the co-stimulatory molecule ICOS. NKT-like cells of pregnant women with COVID-19 up-regulated the expression of IFN-γ, CD107a and Ki67. Meanwhile, we found that ICOSL expression was significantly increased on pDCs in pregnant women with COVID-19. Blocking ICOS in vitro significantly decreased the antiviral activity of NKT-like cells in COVID-19 positive pregnant women, suggesting that ICOS-ICOSL may play an important role in the virus clearance by NKT-like cells. Conclusions: During SARS-CoV-2 infection, NKT-like cells of pregnant women activated through ICOS-ICOSL pathway and played an important role in the antiviral response.
Assuntos
COVID-19 , Ligante Coestimulador de Linfócitos T Induzíveis , Proteína Coestimuladora de Linfócitos T Induzíveis , Células T Matadoras Naturais , Complicações Infecciosas na Gravidez , SARS-CoV-2 , Humanos , Feminino , Gravidez , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , COVID-19/imunologia , COVID-19/virologia , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Adulto , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , SARS-CoV-2/imunologia , Ativação Linfocitária/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Interferon gama/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Lectinas Tipo C/metabolismoRESUMO
Obesity and related diseases have reached epidemic proportions and continue to rise. Beyond creating an economical burden, obesity and its co-morbidities are associated with shortened human life expectancy. Despite major advances, the underlying mechanisms of obesity remain not fully elucidated. Recently, several studies have highlighted that various immune cells are metabolically reprogrammed in obesity, thereby profoundly affecting the immune system. This sheds light on a new field of interest: the impact of obesity-related systemic metabolic changes affecting immune system that could lead to immunosurveillance loss. Among immune cells altered by obesity, invariant Natural Killer T (iNKT) cells have recently garnered intense focus due to their ability to recognize lipid antigen. While iNKT cells are well-described to be affected by obesity, how and to what extent immunometabolic factors (e.g., lipids, glucose, cytokines, adipokines, insulin and free fatty acids) can drive iNKT cells alterations remains unclear, but represent an emerging field of research. Here, we review the current knowledge on iNKT cells in obesity and discuss the immunometabolic factors that could modulate their phenotype and activity.
Assuntos
Células T Matadoras Naturais , Obesidade , Humanos , Obesidade/metabolismo , Obesidade/imunologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Animais , Citocinas/metabolismo , Citocinas/imunologia , Adipocinas/metabolismo , Adipocinas/imunologiaRESUMO
T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim-4) is an immune checkpoint molecule, which involves in numerous inflammatory diseases. Tim-4 is mainly expressed on antigen-presenting cells. However, increasing evidence has shown that Tim-4 is also expressed on natural killer T (NKT) cells. The role of Tim-4 in maintaining NKT cell homeostasis and function remains unknown. In this study, we explored the effect of Tim-4 on NKT cells in acute liver injury. This study found that Tim-4 expression on hepatic NKT cells was elevated during acute liver injury. Tim-4 deficiency enhanced IFN-γ, TNF-α expression while impaired IL-4 production in NKT cells. Loss of Tim-4 drove NKT-cell effector lineages to be skewed to NKT1 subset. Furthermore, Tim-4 KO mice were more susceptible to α-Galactosylceramide (α-GalCer) challenge. In conclusion, our findings indicate that Tim-4 plays an important role in regulating homeostasis and function of NKT cells in acute liver injury. Therefore, Tim-4 might become a new regulator of NKT cells and a potential target for the therapy of acute hepatitis.
Assuntos
Homeostase , Camundongos Knockout , Células T Matadoras Naturais , Animais , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Camundongos , Homeostase/imunologia , Galactosilceramidas/farmacologia , Camundongos Endogâmicos C57BL , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Interleucina-4/metabolismo , Interleucina-4/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/imunologia , MasculinoRESUMO
Invariant natural killer T cell (iNKT) cells produce large amounts of cytokines in response to α-Galactosylceramide (α-GalCer) stimulation. An analog containing two phenyl rings on the acyl chain, C34, was previously found to be more Th1-biased than α-GalCer and triggered greater anticancer activities against breast cancer, melanoma and lung cancer in mice. Since liver is enriched in iNKT cells, we investigated anticancer efficacy of C34 on neuroblastoma with hepatic metastasis. C34 induced Th1-biased cytokine secretions in the liver, significantly suppressed neuroblastoma growth/metastasis and prolonged mouse survival. The anti-tumor efficacy might be attributed to greater expansions of hepatic NKT, NK, CD4+ T, and CD8+ T cells as well as reduction of the number of SSCloGr1intCD11b+ subset of myeloid-derived suppressor cells (MDSCs) in the liver of tumor-bearing mice, as compared to DMSO control group. C34 also upregulated expression of CD1d and CD11c, especially in the SSCloGr1intCD11b+ subset of MDSCs, which might be killed by C34-activated NKT cells, attributing to their reduced number. In addition, C34 also induced expansion of CD4+ T, CD8+ T, and NK cells, which might eliminate neuroblastoma cells. These immune-modulating effects of C34 might act in concert in the local milieu of liver to suppress the tumor growth. Further analysis of database of neuroblastoma revealed that patients with high CD11c expression in the monocytic MDSCs in the tumor had longer survival, suggesting the potential clinical application of C34 for treatment of neuroblastoma.
Assuntos
Glicolipídeos , Neoplasias Hepáticas , Células T Matadoras Naturais , Neuroblastoma , Animais , Neuroblastoma/patologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/imunologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Linhagem Celular Tumoral , Camundongos , Glicolipídeos/farmacologia , Humanos , Feminino , Citocinas/metabolismo , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Antineoplásicos/farmacologia , Galactosilceramidas/farmacologiaRESUMO
Synthetic α-galactosylceramide (αGalCer) and its analogues as powerful agonists for natural killer T (NKT) cell manipulation have received significant attention in immunotherapy and adjuvant development. However, identifying new potent NKT cell agonists, especially those with Th1 selectivity that promote anticancer effects, remains a challenging task. In this work, we introduced a sulfonamide group into the acyl chain of αGalCer to form additional hydrogen bonds to intensify the glycolipid/CD1d interaction. Two compounds GCS-11 and GCS-12 demonstrated remarkable potency while exhibiting different cytokine induction patterns. Compared to αGalCer, the Th1-biased GCS-11 exhibited a 6-fold increase in IFN-γ but not IL-4, while the Th1/2-balanced GCS-12 elicited 7- and 5-fold increase in IFN-γ and IL-4, respectively, in vivo. These findings place them among the most potent NKT cell agonists, with superior antitumor effects. Therefore, hydrogen-bond-involved derivatization could be a powerful strategy to develop potent and polarized NKT cell agonists for various immunotherapies.
Assuntos
Antígenos CD1d , Citocinas , Desenho de Fármacos , Galactosilceramidas , Ligação de Hidrogênio , Células T Matadoras Naturais , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/metabolismo , Animais , Galactosilceramidas/química , Galactosilceramidas/farmacologia , Galactosilceramidas/síntese química , Camundongos , Citocinas/metabolismo , Antígenos CD1d/metabolismo , Antígenos CD1d/química , Humanos , Interleucina-4/metabolismo , Relação Estrutura-Atividade , Camundongos Endogâmicos C57BL , Interferon gama/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonamidas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Linhagem Celular TumoralRESUMO
Persistent human papillomavirus infection is associated with the development of premalignant lesions that can eventually lead to cervical cancer. In this study, we evaluated the expression of activating (NKG2D, DNAM-1) and inhibitory immune checkpoints receptors (PD-1, TIGIT, and Tim-3) in peripheral blood NKT-like (CD3+CD56+) lymphocytes from patients with cervical carcinoma (CC, n = 19), high-grade lesions (HG, n = 8), low-grade lesions (LG, n = 19) and healthy donors (HD, n = 17) using multiparametric flow cytometry. Dimensional data analysis showed four clusters within the CD3+CD56+ cells with different patterns of receptor expression. We observed upregulation of CD16 in CC and HG patients in one of the clusters. In another, TIGIT was upregulated, while DNAM-1 was downregulated. Throughout manual gating, we observed that NKT-like cells expressing activating receptors also co-express inhibitory receptors (PD-1 and TIGIT), which can affect the activation of these cells. A deeper characterization of the functional state of the cells may help to clarify their role in cervical cancer, as will the characterization of the NKT-like cells as cytotoxic CD8+ T cells or members of type I or type II NKT cells.
Assuntos
Antígenos de Diferenciação de Linfócitos T , Antígeno CD56 , Receptor Celular 2 do Vírus da Hepatite A , Células T Matadoras Naturais , Receptor de Morte Celular Programada 1 , Receptores Imunológicos , Neoplasias do Colo do Útero , Humanos , Feminino , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/metabolismo , Receptores Imunológicos/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Adulto , Pessoa de Meia-Idade , Antígeno CD56/metabolismo , Complexo CD3/metabolismo , Lesões Pré-Cancerosas/imunologia , Proteínas de Checkpoint Imunológico/metabolismo , Idoso , Subfamília K de Receptores Semelhantes a Lectina de Células NKRESUMO
Metformin, a medication known for its anti-glycemic properties, also demonstrates potent immune system activation. In our study, using a 4T1 breast cancer model in BALB/C WT mice, we examined metformin's impact on the functional phenotype of multiple immune cells, with a specific emphasis on natural killer T (NKT) cells due to their understudied role in this context. Metformin administration delayed the appearance and growth of carcinoma. Furthermore, metformin increased the percentage of IFN-γ+ NKT cells, and enhanced CD107a expression, as measured by MFI, while decreasing PD-1+, FoxP3+, and IL-10+ NKT cells in spleens of metformin-treated mice. In primary tumors, metformin increased the percentage of NKp46+ NKT cells and increased FasL expression, while lowering the percentages of FoxP3+, PD-1+, and IL-10-producing NKT cells and KLRG1 expression. Activation markers increased, and immunosuppressive markers declined in T cells from both the spleen and tumors. Furthermore, metformin decreased IL-10+ and FoxP3+ Tregs, along with Gr-1+ myeloid-derived suppressor cells (MDSCs) in spleens, and in tumor tissue, it decreased IL-10+ and FoxP3+ Tregs, Gr-1+, NF-κB+, and iNOS+ MDSCs, and iNOS+ dendritic cells (DCs), while increasing the DCs quantity. Additionally, increased expression levels of MIP1a, STAT4, and NFAT in splenocytes were found. These comprehensive findings illustrate metformin's broad immunomodulatory impact across a variety of immune cells, including stimulating NKT cells and T cells, while inhibiting Tregs and MDSCs. This dynamic modulation may potentiate its use in cancer immunotherapy, highlighting its potential to modulate the tumor microenvironment across a spectrum of immune cell types.
Assuntos
Neoplasias da Mama , Metformina , Camundongos Endogâmicos BALB C , Metformina/farmacologia , Metformina/uso terapêutico , Animais , Feminino , Camundongos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Agentes de Imunomodulação/farmacologiaRESUMO
While the transcription factor GATA-3 is well-established for its crucial role in T cell development, its specific influence on invariant natural killer T (iNKT) cells remains relatively unexplored. Using flow cytometry and single-cell transcriptomic analysis, we demonstrated that GATA-3 deficiency in mice leads to the absence of iNKT2 and iNKT17 cell subsets, as well as an altered distribution of iNKT1 cells. Thymic iNKT cells lacking GATA-3 exhibited diminished expression of PLZF and T-bet, key transcription factors involved in iNKT cell differentiation, and reduced production of Th2, Th17, and cytotoxic effector molecules. Single-cell transcriptomics revealed a comprehensive absence of iNKT17 cells, a substantial reduction in iNKT2 cells, and an increase in iNKT1 cells in GATA-3-deficient thymi. Differential expression analysis highlighted the regulatory role of GATA-3 in T cell activation signaling and altered expression of genes critical for iNKT cell differentiation, such as Icos, Cd127, Eomes, and Zbtb16. Notably, restoration of Icos, but not Cd127, expression could rescue iNKT cell development in GATA-3-deficient mice. In conclusion, our study demonstrates the pivotal role of GATA-3 in orchestrating iNKT cell effector lineage differentiation through the regulation of T cell activation pathways and Icos expression, providing insights into the molecular mechanisms governing iNKT cell development and function.
Assuntos
Diferenciação Celular , Linhagem da Célula , Fator de Transcrição GATA3 , Células T Matadoras Naturais , Animais , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/genética , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/metabolismo , Diferenciação Celular/genética , Camundongos , Linhagem da Célula/genética , Camundongos Endogâmicos C57BL , RNA-Seq , Análise de Célula Única , Camundongos Knockout , Análise da Expressão Gênica de Célula ÚnicaRESUMO
BACKGROUND: The increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) incurs substantial morbidity, mortality and healthcare costs. Detection and clinical intervention at early stages of disease improves prognosis; however, we are currently limited by a lack of reliable diagnostic tests for population screening and monitoring responses to therapy. To address this unmet need, we investigated human invariant Natural Killer T cell (iNKT) activation by fat-loaded hepatocytes, leading to the discovery that circulating soluble CD46 (sCD46) levels accurately predict hepatic steatosis. METHODS: sCD46 in plasma was measured using a newly developed immuno-competition assay in two independent cohorts: Prospective living liver donors (n = 156; male = 66, female = 90) and patients with liver tumours (n = 91; male = 58, female = 33). sCD46 levels were statistically evaluated as a predictor of hepatic steatosis. FINDINGS: Interleukin-4-secreting (IL-4+) iNKT cells were over-represented amongst intrahepatic lymphocytes isolated from resected human liver samples. IL-4+ iNKT cells preferentially developed in cocultures with a fat-loaded, hepatocyte-like cell line, HepaRG. This was attributed to induction of matrix metalloproteases (MMP) in fat-loaded HepaRG cells and primary human liver organoids, which led to indiscriminate cleavage of immune receptors. Loss of cell-surface CD46 resulted in unrepressed differentiation of IL-4+ iNKT cells. sCD46 levels were elevated in patients with hepatic steatosis. Discriminatory cut-off values for plasma sCD46 were found that accurately classified patients according to histological steatosis grade. INTERPRETATION: sCD46 is a reliable clinical marker of hepatic steatosis, which can be conveniently and non-invasively measured in serum and plasma samples, raising the possibility of using sCD46 levels as a diagnostic method for detecting or grading hepatic steatosis. FUNDING: F.B. was supported by the Else Kröner Foundation (Award 2016_kolleg.14). G.G. was supported by the Bristol Myers Squibb Foundation for Immuno-Oncology (Award FA-19-009). N.S. was supported by a Wellcome Trust Fellowship (211113/A/18/Z). J.A.H. received funding from the European Union's Horizon 2020 research and innovation programme (Award 860003). J.M.W. received funding from the Else Kröner Foundation (Award 2015_A10).
Assuntos
Biomarcadores , Humanos , Masculino , Biomarcadores/sangue , Feminino , Pessoa de Meia-Idade , Células T Matadoras Naturais/metabolismo , Hepatócitos/metabolismo , Fígado Gorduroso/diagnóstico , Fígado Gorduroso/sangue , Fígado Gorduroso/metabolismo , Adulto , IdosoRESUMO
Trauma rapidly mobilizes the immune response of surrounding tissues and activates regeneration program. Manipulating immune response to promote tissue regeneration shows a broad application prospect. However, the understanding of bone healing dynamics at cellular level remains limited. Here, we characterize the landscape of immune cells after alveolar bone injury and reveal a pivotal role of infiltrating natural killer T (NKT) cells. We observe a rapid increase in NKT cells after injury, which inhibit osteogenic differentiation of mesenchymal stem cells (MSCs) and impair alveolar bone healing. Cxcl2 is up-regulated in NKT cells after injury. Systemic administration of CXCL2-neutralizing antibody or genetic deletion of Cxcl2 improves the bone healing process. In addition, we fabricate a gelatin-based porous hydrogel to deliver NK1.1 depletion antibody, which successfully promotes alveolar bone healing. In summary, our study highlights the importance of NKT cells in the early stage of bone healing and provides a potential therapeutic strategy for accelerating bone regeneration.
Assuntos
Regeneração Óssea , Quimiocina CXCL2 , Células T Matadoras Naturais , Osteogênese , Animais , Camundongos , Regeneração Óssea/genética , Regeneração Óssea/imunologia , Diferenciação Celular , Quimiocina CXCL2/metabolismo , Quimiocina CXCL2/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Osteogênese/genética , Osteogênese/imunologiaRESUMO
Atherosclerosis is an inflammatory disease of blood vessels involving the immune system. Natural killer T (NKT) cells, as crucial components of the innate and acquired immune systems, play critical roles in the development of atherosclerosis. However, the mechanism and clinical relevance of NKT cells in early atherosclerosis are largely unclear. The study investigated the mechanism influencing NKT cell function in apoE deficiency-induced early atherosclerosis. Our findings demonstrated that there were higher populations of NKT cells and interferon-gamma (IFN-γ)-producing NKT cells in the peripheral blood of patients with hyperlipidemia and in the aorta, blood, spleen, and bone marrow of early atherosclerotic mice compared with the control groups. Moreover, we discovered that the infiltration of CD80+ macrophages and CD1d expression on CD80+ macrophages in atherosclerotic mice climbed remarkably. CD1d expression increased in CD80+ macrophages stimulated by oxidized low-density lipoprotein (ox-LDL) ex vivo and in vitro. Ex vivo coculture of macrophages with NKT cells revealed that ox-LDL-induced CD80+ macrophages presented lipid antigen α-Galcer (alpha-galactosylceramide) to NKT cells via CD1d, enabling NKT cells to express more IFN-γ. Furthermore, a greater proportion of CD1d+ monocytes and CD1d+CD80+ monocytes were found in peripheral blood of hyperlipidemic patients compared with that of healthy donors. Positive correlations were found between CD1d+CD80+ monocytes and NKT cells or IFN-γ+ NKT cells in hyperlipidemic patients. Our findings illustrated that CD80+ macrophages stimulated NKT cells to secrete IFN-γ via CD1d-presenting α-Galcer, which may accelerate the progression of early atherosclerosis. Inhibiting lipid antigen presentation by CD80+ macrophages to NKT cells may be a promising immune target for the treatment of early atherosclerosis.NEW & NOTEWORTHY This work proposed the ox-LDL-CD80+ monocyte/macrophage-CD1d-NKT cell-IFN-γ axis in the progression of atherosclerosis. The proinflammatory IFN-γ+ NKT cells are closely related to CD1d+CD80+ monocytes in hyperlipidemic patients. Inhibiting CD80+ macrophages to present lipid antigens to NKT cells through CD1d blocking may be a new therapeutic target for atherosclerosis.
Assuntos
Antígenos CD1d , Aterosclerose , Antígeno B7-1 , Hiperlipidemias , Lipoproteínas LDL , Macrófagos , Células T Matadoras Naturais , Animais , Humanos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Antígenos CD1d/metabolismo , Antígenos CD1d/imunologia , Antígenos CD1d/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Hiperlipidemias/imunologia , Hiperlipidemias/metabolismo , Lipoproteínas LDL/imunologia , Lipoproteínas LDL/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Antígeno B7-1/metabolismo , Antígeno B7-1/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia , Camundongos Endogâmicos C57BL , Feminino , Pessoa de Meia-IdadeRESUMO
The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.
Assuntos
Células-Tronco Hematopoéticas , Imunoterapia Adotiva , Células T Matadoras Naturais , Receptores de Antígenos Quiméricos , Humanos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Animais , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Imunoterapia Adotiva/métodos , Camundongos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Edição de Genes , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/terapia , Neoplasias/imunologia , Linhagem Celular Tumoral , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
How T-cell receptor (TCR) characteristics determine subset commitment during T-cell development is still unclear. Here, we addressed this question for innate-like T cells, mucosal-associated invariant T (MAIT) cells, and invariant natural killer T (iNKT) cells. MAIT and iNKT cells have similar developmental paths, leading in mice to two effector subsets, cytotoxic (MAIT1/iNKT1) and IL17-secreting (MAIT17/iNKT17). For iNKT1 vs iNKT17 fate choice, an instructive role for TCR affinity was proposed but recent data argue against this model. Herein, we examined TCR role in MAIT and iNKT subset commitment through scRNAseq and TCR repertoire analysis. In our dataset of thymic MAIT cells, we found pairs of T-cell clones with identical amino acid TCR sequences originating from distinct precursors, one of which committed to MAIT1 and the other to MAIT17 fates. Quantitative in silico simulations indicated that the number of such cases is best explained by lineage choice being independent of TCR characteristics. Comparison of TCR features of MAIT1 and MAIT17 clonotypes demonstrated that the subsets cannot be distinguished based on TCR sequence. To pinpoint the developmental stage associated with MAIT sublineage choice, we demonstrated that proliferation takes place both before and after MAIT fate commitment. Altogether, we propose a model of MAIT cell development in which noncommitted, intermediate-stage MAIT cells undergo a first round of proliferation, followed by TCR characteristics-independent commitment to MAIT1 or MAIT17 lineage, followed by an additional round of proliferation. Reanalyzing a published iNKT TCR dataset, we showed that this model is also relevant for iNKT cell development.