RESUMO
Introduction: Cytokine release syndrome (CRS) is one of the leading causes of mortality in patients with COVID-19 caused by the SARS-CoV-2 coronavirus. However, the mechanism of CRS induced by SARS-CoV-2 is vague. Methods: Using spike protein combined with IL-2, IFN-γ, and TNF-α to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete CRS-related cytokines, the content of cytokines in the supernatant was detected, and the effects of NK, T, and monocytes were analyzed. Results: This study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more interleukin-2 (IL-2,) which subsequently cooperates with spike protein to facilitate PBMCs to release IL-1ß, IL-6, and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), as well as T cells to release IFN-γ Mechanistically, IFN-γ and TNF-α enhance the transcription of CD40, and the interaction of CD40 and its ligand stabilizes the membrane expression of toll-like receptor 4 (TLR4) that serves as a receptor of spike protein on the surface of monocytes. As a result, there is a constant interaction between spike protein and TLR4, leading to continuous activation of nuclear factor-κ-gene binding (NF-κB). Furthermore, TNF-α also activates NF-κB signaling in monocytes, which further cooperates with IFN-γ and spike protein to modulate NF-κB-dependent transcription of CRS-related inflammatory cytokines. Discussion: Targeting TNF-α/IFN-γ in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19.
Assuntos
COVID-19 , Síndrome da Liberação de Citocina , Interleucina-2 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Linfócitos T , Humanos , Glicoproteína da Espícula de Coronavírus/imunologia , Interleucina-2/metabolismo , Interleucina-2/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Síndrome da Liberação de Citocina/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interferon gama/metabolismo , Interferon gama/imunologia , Receptor 4 Toll-Like/metabolismo , NF-kappa B/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Citocinas/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Over the years, CAR-T cell therapy has achieved remarkable success in treating hematological malignancies. However, this efficacy has not been replicated in the context of glioblastoma (GBM). In this study, a PD1CD28 chimeric molecule was introduced into EGFRvâ ¢-directed CAR-T cells, generating EGFRvâ ¢-P2A-PD1CD28 CAR-T cells. Notably, this modification significantly increased IL-2 secretion and enhanced antigen-dependent activation of CAR-T cells, especially when programmed cell death ligand 1 (PD-L1) was present in vitro. In addition, the in vivo xenograft experiments revealed that the PD1CD28 chimeric molecule played a pivotal role in reducing recurrence rates, effectively controlling recurrent tumor volume, and ultimately prolonging the survival of mice. Collectively, these findings suggest that EGFRvâ ¢-directed CAR-T cells co-expressing the PD1CD28 chimeric molecule have the potential to significantly enhance the treatment efficacy against GBM.
Assuntos
Antígeno B7-H1 , Receptores ErbB , Glioblastoma , Imunoterapia Adotiva , Receptor de Morte Celular Programada 1 , Receptores de Antígenos Quiméricos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Humanos , Camundongos , Glioblastoma/terapia , Glioblastoma/imunologia , Imunoterapia Adotiva/métodos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores ErbB/metabolismo , Receptores ErbB/imunologia , Linhagem Celular Tumoral , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/imunologia , Interleucina-2/metabolismoRESUMO
Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.
Assuntos
Linfócitos T CD8-Positivos , Citoesqueleto , Interleucina-2 , Camundongos Endogâmicos C57BL , Fator de Resposta Sérica , Transativadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/metabolismo , Interleucina-2/genética , Animais , Transativadores/metabolismo , Transativadores/genética , Citoesqueleto/metabolismo , Camundongos , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/genética , Listeriose/microbiologia , Actinas/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Camundongos Knockout , Proliferação de Células , Ativação LinfocitáriaRESUMO
Genome-wide association studies (GWAS) have identified hundreds of genetic signals associated with autoimmune disease. The majority of these signals are located in non-coding regions and likely impact cis-regulatory elements (cRE). Because cRE function is dynamic across cell types and states, profiling the epigenetic status of cRE across physiological processes is necessary to characterize the molecular mechanisms by which autoimmune variants contribute to disease risk. We localized risk variants from 15 autoimmune GWAS to cRE active during TCR-CD28 co-stimulation of naïve human CD4+ T cells. To characterize how dynamic changes in gene expression correlate with cRE activity, we measured transcript levels, chromatin accessibility, and promoter-cRE contacts across three phases of naive CD4+ T cell activation using RNA-seq, ATAC-seq, and HiC. We identified ~1200 protein-coding genes physically connected to accessible disease-associated variants at 423 GWAS signals, at least one-third of which are dynamically regulated by activation. From these maps, we functionally validated a novel stretch of evolutionarily conserved intergenic enhancers whose activity is required for activation-induced IL2 gene expression in human and mouse, and is influenced by autoimmune-associated genetic variation. The set of genes implicated by this approach are enriched for genes controlling CD4+ T cell function and genes involved in human inborn errors of immunity, and we pharmacologically validated eight implicated genes as novel regulators of T cell activation. These studies directly show how autoimmune variants and the genes they regulate influence processes involved in CD4+ T cell proliferation and activation.
Assuntos
Linfócitos T CD4-Positivos , Cromatina , Estudo de Associação Genômica Ampla , Interleucina-2 , Ativação Linfocitária , Humanos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cromatina/metabolismo , Cromatina/genética , Ativação Linfocitária/genética , Interleucina-2/genética , Interleucina-2/metabolismo , Animais , Camundongos , Elementos Facilitadores Genéticos/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Regulação da Expressão Gênica , Autoimunidade/genéticaRESUMO
The thymus is the central organ involved with T-cell development and the production of naïve T cells. During normal aging, the thymus undergoes marked involution, reducing naïve T-cell output and resulting in a predominance of long-lived memory T cells in the periphery. Outside of aging, systemic stress responses that induce corticosteroids (CS), or other insults such as radiation exposure, induce thymocyte apoptosis, resulting in a transient acute thymic involution with subsequent recovery occurring after cessation of the stimulus. Despite the increasing utilization of immunostimulatory regimens in cancer, effects on the thymus and naïve T cell output have not been well characterized. Using both mouse and human systems, the thymic effects of systemic immunostimulatory regimens, such as high dose IL-2 (HD IL-2) with or without agonistic anti-CD40 mAbs and acute primary viral infection, were investigated. These regimens produced a marked acute thymic involution in mice, which correlated with elevated serum glucocorticoid levels and a diminishment of naïve T cells in the periphery. This effect was transient and followed with a rapid thymic "rebound" effect, in which an even greater quantity of thymocytes was observed compared to controls. Similar results were observed in humans, as patients receiving HD IL-2 treatment for cancer demonstrated significantly increased cortisol levels, accompanied by decreased peripheral blood naïve T cells and reduced T-cell receptor excision circles (TRECs), a marker indicative of recent thymic emigrants. Mice adrenalectomized prior to receiving immunotherapy or viral infection demonstrated protection from this glucocorticoid-mediated thymic involution, despite experiencing a substantially higher inflammatory cytokine response and increased immunopathology. Investigation into the effects of immunostimulation on middle aged (7-12 months) and advance aged (22-24 months) mice, which had already undergone significant thymic involution and had a diminished naïve T cell population in the periphery at baseline, revealed that even further involution was incurred. Thymic rebound hyperplasia, however, only occurred in young and middle-aged recipients, while advance aged not only lacked this rebound hyperplasia, but were entirely absent of any indication of thymic restoration. This coincided with prolonged deficits in naïve T cell numbers in advanced aged recipients, further skewing the already memory dominant T cell pool. These results demonstrate that, in both mice and humans, systemic immunostimulatory cancer therapies, as well as immune challenges like subacute viral infections, have the potential to induce profound, but transient, glucocorticoid-mediated thymic involution and substantially reduced thymic output, resulting in the reduction of peripheral naive T cells. This can then be followed by a marked rebound effect with naïve T cell restoration, events that were shown not to occur in advanced-aged mice.
Assuntos
Glucocorticoides , Timo , Animais , Timo/imunologia , Timo/efeitos dos fármacos , Camundongos , Humanos , Glucocorticoides/uso terapêutico , Glucocorticoides/farmacologia , Feminino , Masculino , Idoso , Envelhecimento/imunologia , Pessoa de Meia-Idade , Interleucina-2/metabolismo , Adulto , Timócitos/imunologia , Timócitos/metabolismo , Hiperplasia do Timo/imunologia , Camundongos Endogâmicos C57BL , Imunização , HiperplasiaRESUMO
CD38 has emerged as a potential therapeutic target for patients with systemic lupus erythematosus (SLE) but it is not known whether CD38 alters CD4+ T cell function. Using primary human T cells and CD38-sufficient and CD38-deficient Jurkat T cells, we demonstrate that CD38 shifts the T cell lipid profile of gangliosides from GM3 to GM2 by upregulating B4GALNT1 in a Sirtuin 1-dependent manner. Enhanced expression of GM2 causes ER stress by enhancing Ca2+ flux through the PLCγ1-IP3 pathway. Interestingly, correction of the calcium overload by an IP3 receptor inhibitor, but not by a store-operated calcium entry (SOCE) inhibitor, improves IL-2 production by CD4+ T cells in SLE. This study demonstrates that CD38 affects calcium homeostasis in CD4+ T cells by controlling cell membrane lipid composition that results in suppressed IL-2 production. CD38 inhibition with biologics or small drugs should be expected to benefit patients with SLE.
Assuntos
ADP-Ribosil Ciclase 1 , Linfócitos T CD4-Positivos , Cálcio , Membrana Celular , Interleucina-2 , Lúpus Eritematoso Sistêmico , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/genética , Interleucina-2/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , ADP-Ribosil Ciclase 1/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Cálcio/metabolismo , Células Jurkat , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Fosfolipase C gama/metabolismo , Fosfolipase C gama/genética , FemininoRESUMO
Regulatory T cells (Treg) are critical players of immune tolerance that develop in the thymus via two distinct developmental pathways involving CD25+Foxp3- and CD25-Foxp3lo precursors. However, the mechanisms regulating the recently identified Foxp3lo precursor pathway remain unclear. Here, we find that the membrane-bound lymphotoxin α1ß2 (LTα1ß2) heterocomplex is upregulated during Treg development upon TCR/CD28 and IL-2 stimulation. We show that Lta expression limits the maturational development of Treg from Foxp3lo precursors by regulating their proliferation, survival, and metabolic profile. Transgenic reporter mice and transcriptomic analyses further reveal that medullary thymic epithelial cells (mTEC) constitute an unexpected source of IL-4. We demonstrate that LTα1ß2-lymphotoxin ß receptor-mediated interactions with mTEC limit Treg development by down-regulating IL-4 expression in mTEC. Collectively, our findings identify the lymphotoxin axis as the first inhibitory checkpoint of thymic Treg development that fine-tunes the Foxp3lo Treg precursor pathway by limiting IL-4 availability.
Assuntos
Fatores de Transcrição Forkhead , Interleucina-4 , Receptor beta de Linfotoxina , Linfotoxina-alfa , Transdução de Sinais , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Interleucina-4/metabolismo , Camundongos , Linfotoxina-alfa/metabolismo , Linfotoxina-alfa/genética , Receptor beta de Linfotoxina/metabolismo , Receptor beta de Linfotoxina/genética , Timo/imunologia , Timo/citologia , Timo/metabolismo , Células Epiteliais/metabolismo , Camundongos Endogâmicos C57BL , Diferenciação Celular , Camundongos Transgênicos , Interleucina-2/metabolismo , Proliferação de Células , Heterotrímero de Linfotoxina alfa1 e beta2/metabolismo , Heterotrímero de Linfotoxina alfa1 e beta2/genéticaRESUMO
We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors.
Assuntos
Interferon gama , Interleucina-2 , Receptor de Interferon alfa e beta , Linfócitos T , Humanos , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/genética , Linfócitos T/metabolismo , Linfócitos T/imunologia , Interleucina-2/metabolismo , Interferon gama/metabolismo , Receptores de Interleucina-2/metabolismo , Receptores de Interleucina-2/genética , Ligação Proteica , Ativação Linfocitária , Células HEK293RESUMO
Cytokine-mediated STAT5 protein activation is vital for lymphocyte development and function. In vitro tyrosine phosphorylation of a C-terminal tyrosine is critical for activation of STAT5A and STAT5B; however, the importance of STAT5 tyrosine phosphorylation in vivo has not been assessed. Here we generate Stat5a and Stat5b tyrosine-to-phenylalanine mutant knockin mice and find they have greatly reduced CD8+ T-cell numbers and profoundly diminished IL-2-induced proliferation of these cells, and this correlates with reduced induction of Myc, pRB, a range of cyclins and CDKs, and a partial G1âS phase-transition block. These mutant CD8+ T cells also exhibit decreased IL-2-mediated activation of pERK and pAKT, which we attribute in part to diminished expression of IL-2Rß and IL-2Rγ. Our findings thus demonstrate that tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling. Moreover, our transcriptomic and proteomic analyses elucidate the molecular basis of the IL-2-induced proliferation of CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos , Proliferação de Células , Interleucina-2 , Fator de Transcrição STAT5 , Transdução de Sinais , Tirosina , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT5/genética , Animais , Interleucina-2/metabolismo , Fosforilação , Tirosina/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Camundongos , Subunidade beta de Receptor de Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Camundongos Endogâmicos C57BL , Técnicas de Introdução de Genes , Ativação LinfocitáriaRESUMO
IL-32 expression is important for pathogen clearance but detrimental in chronic inflammation, autoimmunity, and cancer. T cells are major IL-32 producers in these diseases and key mediators of pathogen and tumor elimination but also autoimmune destruction. However, their contribution to IL-32 biology during immune responses is hardly understood due to several isoforms with divergent inflammatory properties. Here, we identified IL-32ß as the predominant isoform in various T cell subsets of healthy individuals and breast cancer patients with the highest levels detected in intratumoral regulatory T cells. We show that IL-32ß is induced by IL-2 but IL-32ß release requires T Cell Receptor rather than IL2R stimulation. Using inhibitors of protein secretion pathways and serial (ultra)centrifugation of T cell supernatants, we demonstrate that T cells actively secrete IL-32ß unconventionally, as a free protein and, to a minor degree, through exosomes. Thus, our data identify activated T cells as major IL-32ß secretors in health and cancer.
Assuntos
Neoplasias da Mama , Interleucina-2 , Interleucinas , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T , Humanos , Interleucinas/metabolismo , Interleucina-2/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Feminino , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoAssuntos
Adenoviridae , Interleucina-2 , Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias Ovarianas , Humanos , Neoplasias Ovarianas/terapia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/genética , Feminino , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Adenoviridae/genética , Interleucina-2/genética , Interleucina-2/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Animais , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Terapia Genética/métodosRESUMO
BACKGROUND: Cannabidiol (CBD), the major non-psychoactive component of cannabis, exhibits anti-inflammatory properties, but less is known about the immunomodulatory potential of CBD on activated natural killer (NK) cells and/or their targets. Many tumor cells present heat shock protein 70 (Hsp70) on their cell surface in a tumor-specific manner and although a membrane Hsp70 (mHsp70) positive phenotype serves as a target for Hsp70-activated NK cells, a high mHsp70 expression is associated with tumor aggressiveness. This study investigated the immuno-modulatory potential of CBD on NK cells stimulated with TKD Hsp70 peptide and IL-2 (TKD+IL-2) and also on HCT116 p53wt and HCT116 p53-/- colorectal cancer cells exhibiting high and low basal levels of mHsp70 expression. RESULTS: Apart from an increase in the density of NTB-A and a reduced expression of LAMP-1, the expression of all other activatory NK cell receptors including NKp30, NKG2D and CD69 which are significantly up-regulated after stimulation with TKD+IL-2 remained unaffected after a co-treatment with CBD. However, the release of major pro-inflammatory cytokines by NK cells such as interferon-γ (IFN-γ) and the effector molecule granzyme B (GrzB) was significantly reduced upon CBD treatment. With respect to the tumor target cells, CBD significantly reduced the elevated expression of mHsp70 but had no effect on the low basal mHsp70 expression. Expression of other NK cell ligands such as MICA and MICB remained unaffected, and the NK cell ligands ULBP and B7-H6 were not expressed on these target cells. Consistent with the reduced mHsp70 expression, treatment of both effector and target cells with CBD reduced the killing of high mHsp70 expressing tumor cells by TKD+IL-2+CBD pre-treated NK cells but had no effect on the killing of low mHsp70 expressing tumor cells. Concomitantly, CBD treatment reduced the TKD+IL-2 induced increased release of IFN-γ, IL-4, TNF-α and GrzB, but CBD had no effect on the release of IFN-α when NK cells were co-incubated with tumor target cells. CONCLUSION: Cannabidiol (CBD) may potentially diminish the anti-tumor effectiveness of TKD+IL-2 activated natural killer (NK) cells.
Assuntos
Canabidiol , Proteínas de Choque Térmico HSP70 , Células Matadoras Naturais , Ativação Linfocitária , Humanos , Canabidiol/farmacologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Células HCT116 , Fatores Imunológicos/farmacologia , Interleucina-2/metabolismo , Interleucina-2/imunologia , Granzimas/metabolismo , Interferon gama/metabolismo , Interferon gama/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacosRESUMO
BACKGROUND: Lytic Epstein-Barr virus (EBV) infection plays a major role in the pathogenesis of nasopharyngeal carcinoma (NPC). For patients with recurrent or metastatic NPC and resistant to conventional therapies, adoptive cell therapy using EBV-specific cytotoxic T cells (EBV-CTLs) is a promising option. However, the long production period (around 3 to 4 weeks) and low EBV-CTL purity (approximately 40% of total CD8 T cells) in the cell product limits the application of EBV-CTLs in clinics. Thus, this study aimed to establish a protocol for the rapid production of EBV-CTLs. METHODS: By culturing peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors with EBV-specific peptides and interleukin (IL)-2, IL-15, and interferon α (IFN-α) for 9 days, we identified that IL-15 can enhance IL-2-mediated CTL activation and significantly increase the yield of CTLs. RESULTS: When IFN-α was used in IL-2/IL-15-mediated CTL production from days 0 to 6, the productivity of EBV-CTLs and EBV-specific cytotoxicity significantly were reinforced relative to EBV-CTLs from IL-2/IL-15 treatment. Additionally, IFN-α-induced production improvement of virus-specific CTLs was not only the case for EBV-CTLs but also for cytomegalovirus-specific CTLs. CONCLUSION: We established a novel protocol to rapidly expand highly pure EBV-CTLs from PBMCs, which can produce EBV-CTLs in 9 days and does not require feeder cells during cultivation.
Assuntos
Herpesvirus Humano 4 , Linfócitos T Citotóxicos , Humanos , Linfócitos T Citotóxicos/imunologia , Herpesvirus Humano 4/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Interleucina-15/metabolismo , Interferon-alfa/metabolismo , Citotoxicidade Imunológica , Carcinoma Nasofaríngeo/virologia , Carcinoma Nasofaríngeo/imunologia , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/imunologia , Neoplasias Nasofaríngeas/virologia , Neoplasias Nasofaríngeas/patologia , Ativação Linfocitária/imunologia , Imunoterapia Adotiva/métodosRESUMO
Sepsis, a life-threatening condition, involves complex interactions among metabolic alterations, inflammatory mediators, and host responses. This study utilized a bidirectional Mendelian randomization approach to investigate the causal relationships between 1400 metabolites and sepsis, and the mediating role of inflammatory factors. We identified 36 metabolites significantly associated with sepsis (p < 0.05), with AXIN1, FGF-19, FGF-23, IL-4, and OSM showing an inverse association, suggesting a protective role, while IL-2 exhibited a positive correlation, indicating a potential risk factor. Among these metabolites, Piperine and 9-Hydroxystearate demonstrated particularly interesting protective effects against sepsis. Piperine's protective effect was mediated through its interaction with AXIN1, contributing to a 16.296% reduction in sepsis risk. This suggests a potential pathway where Piperine influences sepsis outcomes by modulating AXIN1 levels. 9-Hydroxystearate also exhibited a protective role against sepsis, mediated through its positive association with FGF-19 and negative association with IL-2, contributing 9.436% and 12.565%, respectively, to its protective effect. Experimental validation confirmed significantly elevated IL-2 levels and reduced FGF-19, AXIN1, piperine, and 9-hydroxyoctadecanoic acid levels in sepsis patients compared to healthy controls. Piperine levels positively correlated with AXIN1, while 9-hydroxyoctadecanoic acid levels negatively correlated with IL-2 and positively correlated with FGF-19, supporting the Mendelian randomization findings. Our findings provide insights into the molecular mechanisms of sepsis, highlighting the unique roles and contributions of specific metabolites and their interactions with inflammatory mediators. This study enhances our understanding of sepsis pathophysiology and opens avenues for targeted therapeutic interventions and biomarker development for sepsis management. However, further research is essential to validate these pathways across diverse populations and fully explore the roles of these metabolites in sepsis.
Assuntos
Alcaloides , Proteína Axina , Fatores de Crescimento de Fibroblastos , Análise da Randomização Mendeliana , Alcamidas Poli-Insaturadas , Sepse , Humanos , Sepse/metabolismo , Sepse/genética , Alcamidas Poli-Insaturadas/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Proteína Axina/metabolismo , Proteína Axina/genética , Piperidinas/uso terapêutico , Benzodioxóis , Inflamação/metabolismo , Interleucina-2/metabolismo , Interleucina-2/genética , Mediadores da Inflamação/metabolismo , Fator de Crescimento de Fibroblastos 23RESUMO
IL-2 regulates the immune response by interacting with different IL-2 receptor (IL-2R) subunits. High dose of IL-2 binds to IL-2Rßγc heterodimer, which induce various side effects while activating immune function. Disrupting IL-2 and IL-2R interactions can block IL-2 mediated immune response. Here, we used a computational approach to de novo design mini-binder proteins against IL-2R ß chain (IL-2Rß) to block IL-2 signaling. The hydrophobic region where IL-2 binds to IL-2Rß was selected and the promising binding mode was broadly explored. Three mini-binders with amino acid numbers ranging from 55 to 65 were obtained and binder 1 showed the best effects in inhibiting CTLL-2 cells proliferation and STAT5 phosphorylation. Molecular dynamics simulation showed that the binding of binder 1 to IL-2Rß was stable; the free energy of binder1/IL-2Rß complex was lower, indicating that the affinity of binder 1 to IL-2Rß was higher than that of IL-2. Free energy decomposition suggested that the ARG35 and ARG131 of IL-2Rß might be the key to improve the affinity of binder. Our efforts provided new insights in developing of IL-2R blocker, offering a potential strategy for ameliorating the side effects of IL-2 treatment.
Assuntos
Subunidade beta de Receptor de Interleucina-2 , Interleucina-2 , Simulação de Dinâmica Molecular , Ligação Proteica , Subunidade beta de Receptor de Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/química , Interleucina-2/metabolismo , Interleucina-2/química , Humanos , Proliferação de Células/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Fosforilação/efeitos dos fármacos , Animais , Simulação de Acoplamento MolecularRESUMO
BACKGROUND: Interleukin (IL)-2 is a key cytokine capable of modulating the immune response by activating natural killer (NK) cells. This study was recruited to explore the therapeutic potential of IL-2-activated NK-92 cells in endometriosis in vitro. METHODS: Ectopic endometrial stromal cells (EESCs) were isolated and co-cultured with IL-2-activated NK-92 cells at varying effector-to-target (E:T) ratios (1:0 [Control], 1:1, 1:3, and 1:9). The viability, cytotoxicity, and cell surface antigen expression of IL-2-activated NK-92 cells were assessed. The viability, apoptosis, invasion, and migration ability of EESCs co-cultured with NK-92 cells at different ratios were evaluated. The apoptosis-related proteins, invasion and migration-related proteins as well as MEK/ERK pathway were examined via western blot. Each experiment was repeated three times. RESULTS: IL-2 activation enhanced NK-92 cytotoxicity in a concentration-dependent manner. Co-culturing EESCs with IL-2-activated NK-92 cells at E:T ratios of 1:1, 1:3, and 1:9 reduced EESC viability by 20%, 45%, and 70%, respectively, compared to the control group. Apoptosis rates in EESCs increased in correlation with the NK-92 cell proportion, with the highest rate observed at a 1:9 ratio. Moreover, EESC invasion and migration were significantly inhibited by IL-2-activated NK-92 cells, with a 60% reduction in invasion and a 50% decrease in migration at the 1:9 ratio. Besides, the MEK/ERK signalling pathway was down-regulated in EESCs by IL-2-activated NK-92 cells. CONCLUSION: IL-2-activated NK-92 cells exhibit potent cytotoxic effects against EESCs. They promote EESC apoptosis and inhibit viability, invasion, and migration through modulating the MEK/ERK signalling pathway.
Endometriosis is a common chronic systemic disease affecting approximately 190 million women worldwide. However, clinical treatments for endometriosis remain challenging due to the scarcity of high-quality scientific evidence and conflicting available guidelines. This research was designed to explore whether interleukin (IL)-2 affected the progression of endometriosis by modulating endometrial stromal cell apoptosis and natural killer (NK) cell-mediated cytotoxicity, thereby providing new therapeutic methods for endometriosis.
Assuntos
Apoptose , Técnicas de Cocultura , Endometriose , Interleucina-2 , Células Matadoras Naturais , Humanos , Endometriose/patologia , Endometriose/imunologia , Feminino , Interleucina-2/farmacologia , Interleucina-2/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Apoptose/efeitos dos fármacos , Adulto , Endométrio/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Progressão da Doença , Sobrevivência Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células CultivadasRESUMO
Mucosal vaccination strategies are easier to implement than others in large-scale poultry farming. However, the adjuvants that are approved for veterinary use, which are predominantly aluminum- and oil-emulsion-based adjuvants, are not suitable for mucosal vaccination and carry a risk of adverse reactions. In this study, we engineered a novel Lactobacillus plantarum NC8 strain that co-expresses chicken interleukin-2 (IL-2) and IL-17B, which we designated NC8-ChIL2-17B, and evaluated its potential as an oral immunoadjuvant. The immunomodulatory properties of NC8-ChIL2-17B were evidenced by its ability to activate macrophages and inhibit the proliferation of infectious bronchitis virus (IBV) in vitro. We then confirmed its immunoadjuvant activity in vivo by orally administering NC8-ChIL2-17B along with a commercial IBV vaccine to chicks. The results indicated that NC8-ChIL2-17B enhanced the immune response elicited by the IBV vaccine and increased the levels of IBV-specific IgG and sIgA antibodies produced in response to IBV infection. Additionally, administration of NC8-ChIL2-17B promoted weight gain and beneficially modulated the gut microbiota, resulting in improved chicken performance. These findings suggest that oral administration of NC8-ChIL2-17B is a promising strategy to enhance the immune efficacy of the IBV vaccine in chickens, offering an efficacious alternative adjuvant.
Assuntos
Adjuvantes Imunológicos , Galinhas , Infecções por Coronavirus , Vírus da Bronquite Infecciosa , Interleucina-17 , Interleucina-2 , Lactobacillus plantarum , Doenças das Aves Domésticas , Vacinas Virais , Animais , Galinhas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/imunologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Interleucina-2/metabolismo , Interleucina-2/genética , Vírus da Bronquite Infecciosa/imunologia , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Interleucina-17/metabolismo , Administração Oral , Vacinação/veterináriaRESUMO
In previous studies, we developed a novel fusion protein named "melittin-MIL-2" which exhibited more anti-tumor activity. However, it remains unclear whether melittin-MIL-2 possesses antitumor immune effect on lung adenocarcinoma. In this study, the immune effect and mechanism of melittin-MIL-2 inhibiting the growth and invasion of lung adenocarcinoma will be investigated, in order to provide novel perspectives for the immunotherapy of lung cancer. The results indicated that melittin-MIL-2 promoted T cell proliferation, enhanced NK cell cytotoxicity, and boosted IFN-γ secretion in PBMCs. After melittin-MIL-2 stimulation, perforin expression and LAK/NK-like killing activities of human PBMCs and NK cells were significantly enhanced. Melittin-MIL-2 is capable of hampering the development and proliferation of lung adenocarcinoma cell A549. ICAM-1 and Fas expression in A549 cells exposed to melittin-MIL-2 rose significantly. The expression levels of TLR8 and VEGF in A549 cells decreased significantly after melittin-MIL-2 stimulation. In vivo, melittin-MIL-2 substantially impeded the growth of lung adenocarcinoma and formed an immune-stimulating microenvironment locally in tumor tissues. In conclusion, the novel fusion protein melittin-MIL-2 exhibits strong anti-tumor immune effect in lung adenocarcinoma cell A549 via activating the LFA-1/ICAM-1 and Fas/FasL pathways to enhance cytolytic activity, upregulating the secretion of IFN-γ and perforin, and boosting LAK/NK-like killing activities. Immuno-effector cells and their secreted cytokines can form immune stimulation microenvironment locally in lung adenocarcinoma Lewis mice tissue.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Meliteno , Meliteno/farmacologia , Humanos , Animais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Camundongos , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/genética , Proliferação de Células/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Interleucina-2/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/genética , Imunoterapia/métodosRESUMO
Interleukin-2 (IL-2) has been used in cancer treatment for over 30 years. However, due to its high toxicity, new mutant variants have been developed. These variants retain some of the biological properties of the original molecule but offer other therapeutic advantages. At the Center of Molecular Immunology, the IL-2 no-alpha mutein, an IL-2 agonist with lower toxicity than wtIL-2, has been designed, produced, and is currently being evaluated in a Phase I/II clinical trial. The mutein is produced in E. coli as an insoluble material that must be refolded in vitro to yield a fully active protein. Controlled oxidation steps are essential in the purification process of recombinant proteins produced in E. coli to ensure the proper formation of the disulfide bonds in the molecules. In this case, the new purification process includes a copper-catalyzed air oxidation step to induce disulfide bond establishment. The optimal conditions of pH, copper, protein and detergent concentration for this step were determined through screening. The produced protein demonstrated a conserved 3D structure, higher purity, and greater biological activity than the obtained by established process without the oxidation step. Four batches were produced and evaluated, demonstrating the consistency of the new process.
Assuntos
Cobre , Escherichia coli , Interleucina-2 , Oxirredução , Proteínas Recombinantes , Cobre/química , Interleucina-2/metabolismo , Interleucina-2/genética , Escherichia coli/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ar , Redobramento de Proteína , Catálise , Concentração de Íons de HidrogênioRESUMO
OBJECTIVE: To investigate the levels of 12 kinds of cytokines in seminal plasma and their correlations with routine semen parameters. METHODS: The remaining seminal plasma samples of 134 patients undergoing routine semen examination were collected for detecting cytokines. The parameters for sperm concentration, percentage of progressively motile sperm (PR), and motility were analyzed by a computer-assisted sperm analysis (CASA) system. According to the results of sperm concentration, PR and motility, 134 patients were divided into the normal routine semen parameters group, oligoasthenospermia group and azoospermia group. The levels of 12 kinds of cytokines in seminal plasma, including interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17, interferin (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α, were detected by flow cytometry. Two seminal plasma samples were detected for 10 times, respectively, to calculate the coefficients of variation (CV) of each cytokine. The linear range of each cytokine was measured using the standard, and the correlation coefficient (r) was calculated. RESULTS: The r2 of 12 kinds of cytokines detected by flow cytometry were all greater than 0.99. The reproducibility of 2 seminal plasma samples showed that the CVs of all cytokines were lower than 15 % except for TNF-α in sample 1 (15.15 %). Seminal plasma IL-6 levels were negatively correlated with semen volume (P < 0.01). Seminal plasma IL-5 levels were positively correlated with sperm concentration (P < 0.01). Seminal plasma IL-8 levels were negatively correlated with sperm motility (P < 0.01). Seminal plasma IL-8, IL-17 and IL-12P70 levels were negatively correlated with sperm PR (P < 0.05). In addition to the significant negative correlation between IL-5 and IL-17 (P < 0.05), there was a significant positive correlation between the majority of other cytokines. The levels of seminal plasma IL-17 and IL-12P70 in the oligoasthenospermia group and IL-1ß and IL-12P70 in the azoospermia group were significantly higher than those in the normal routine semen parameters group (P ≤ 0.05), while the levels of IL-10 in the azoospermia group were significantly lower than that in the normal routine semen parameters group (P < 0.05). CONCLUSION: There are certain correlations between seminal plasma cytokines and routine semen parameters and strong correlations between different seminal plasma cytokines, suggesting that the imbalance between seminal plasma cytokines may affect sperm quality. However, it still needs to be further confirmed by large samples and multi-center clinical studies and related basic researches.