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1.
Cells ; 10(6)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203839

RESUMO

The bone marrow (BM) is key to protective immunological memory because it harbors a major fraction of the body's plasma cells, memory CD4+ and memory CD8+ T-cells. Despite its paramount significance for the human immune system, many aspects of how the BM enables decade-long immunity against pathogens are still poorly understood. In this review, we discuss the relationship between BM survival niches and long-lasting humoral immunity, how intrinsic and extrinsic factors define memory cell longevity and show that the BM is also capable of adopting many responsibilities of a secondary lymphoid organ. Additionally, with more and more data on the differentiation and maintenance of memory T-cells and plasma cells upon vaccination in humans being reported, we discuss what factors determine the establishment of long-lasting immunological memory in the BM and what we can learn for vaccination technologies and antigen design. Finally, using these insights, we touch on how this holistic understanding of the BM is necessary for the development of modern and efficient vaccines against the pandemic SARS-CoV-2.


Assuntos
Imunidade Adaptativa/fisiologia , Medula Óssea/fisiologia , Plasmócitos/citologia , Linfócitos T/citologia , Vacinologia , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , COVID-19/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Celular/fisiologia , Memória Imunológica/fisiologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Vacinologia/métodos , Vacinologia/tendências
2.
Cells ; 10(6)2021 06 15.
Artigo em Inglês | MEDLINE | ID: covidwho-1273393

RESUMO

The bone marrow (BM) is key to protective immunological memory because it harbors a major fraction of the body's plasma cells, memory CD4+ and memory CD8+ T-cells. Despite its paramount significance for the human immune system, many aspects of how the BM enables decade-long immunity against pathogens are still poorly understood. In this review, we discuss the relationship between BM survival niches and long-lasting humoral immunity, how intrinsic and extrinsic factors define memory cell longevity and show that the BM is also capable of adopting many responsibilities of a secondary lymphoid organ. Additionally, with more and more data on the differentiation and maintenance of memory T-cells and plasma cells upon vaccination in humans being reported, we discuss what factors determine the establishment of long-lasting immunological memory in the BM and what we can learn for vaccination technologies and antigen design. Finally, using these insights, we touch on how this holistic understanding of the BM is necessary for the development of modern and efficient vaccines against the pandemic SARS-CoV-2.


Assuntos
Imunidade Adaptativa/fisiologia , Medula Óssea/fisiologia , Plasmócitos/citologia , Linfócitos T/citologia , Vacinologia , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , COVID-19/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Celular/fisiologia , Memória Imunológica/fisiologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Vacinologia/métodos , Vacinologia/tendências
3.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199417

RESUMO

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite this achievement, mAbs-based therapies are struggling with limitations including poor tissue and tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs, which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1 pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have been reported, their development lags behind the corresponding mAb, partly due to the challenges of developing drug-like small molecules. Herein, we report the discovery of a series of novel inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058 as a starting point. Among them, compound A9 stands out as the most promising candidate with excellent PD-L1 inhibitory activity (IC50 = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1 (KD = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1 interaction and is worthy for further study.


Assuntos
Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/citologia , Antígeno B7-H1/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Interferon gama/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Cultura Primária de Células , Receptor de Morte Celular Programada 1/química , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
4.
Nat Commun ; 12(1): 3684, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140495

RESUMO

Interaction between tumor cells and immune cells in the tumor microenvironment is important in cancer development. Immune cells interact with the tumor cells to shape this process. Here, we use single-cell RNA sequencing analysis to delineate the immune landscape and tumor heterogeneity in a cohort of patients with HBV-associated human hepatocellular carcinoma (HCC). We found that tumor-associated macrophages suppress tumor T cell infiltration and TIGIT-NECTIN2 interaction regulates the immunosuppressive environment. The cell state transition of immune cells towards a more immunosuppressive and exhaustive status exemplifies the overall cancer-promoting immunocellular landscape. Furthermore, the heterogeneity of global molecular profiles reveals co-existence of intra-tumoral and inter-tumoral heterogeneity, but is more apparent in the latter. This analysis of the immunosuppressive landscape and intercellular interactions provides mechanistic information for the design of efficacious immune-oncology treatments in hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/imunologia , Regulação da Expressão Gênica/imunologia , Neoplasias Hepáticas/imunologia , Macrófagos/imunologia , Microambiente Tumoral/imunologia , Algoritmos , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Proliferação de Células , Regulação da Expressão Gênica/genética , Vírus da Hepatite B/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nectinas/genética , Nectinas/metabolismo , Análise de Componente Principal , Prognóstico , RNA-Seq , Receptores Imunológicos/metabolismo , Análise de Célula Única , Linfócitos T/citologia , Linfócitos T/imunologia , Microambiente Tumoral/genética
5.
Nat Commun ; 12(1): 3933, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168132

RESUMO

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.


Assuntos
Epigênese Genética , Células Epiteliais/citologia , Complexo Repressor Polycomb 2/genética , Timo/citologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Epiteliais/fisiologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Linfócitos T/fisiologia , Timócitos/citologia , Timócitos/fisiologia , Timo/fisiologia
6.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072618

RESUMO

Adaptive immunity relies on the V(D)J DNA recombination of immunoglobulin (Ig) and T cell receptor (TCR) genes, which enables the recognition of highly diverse antigens and the elicitation of antigen-specific immune responses. This process is mediated by recombination-activating gene (Rag) 1 and Rag2 (Rag1/2), whose expression is strictly controlled in a cell type-specific manner; the expression of Rag1/2 genes represents a hallmark of lymphoid lineage commitment. Although Rag genes are known to be evolutionally conserved among jawed vertebrates, how Rag genes are regulated by lineage-specific transcription factors (TFs) and how their regulatory system evolved among vertebrates have not been fully elucidated. Here, we reviewed the current body of knowledge concerning the cis-regulatory elements (CREs) of Rag genes and the evolution of the basic helix-loop-helix TF E protein regulating Rag gene CREs, as well as the evolution of the antagonist of this protein, the Id protein. This may help to understand how the adaptive immune system develops along with the evolution of responsible TFs and enhancers.


Assuntos
Imunidade Adaptativa/genética , Elementos Facilitadores Genéticos , Evolução Molecular , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Regulação da Expressão Gênica , Humanos , Sequências Reguladoras de Ácido Nucleico , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Recombinação V(D)J
7.
J Med Chem ; 64(11): 7646-7666, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34037385

RESUMO

Two series of novel o-(biphenyl-3-ylmethoxy)nitrophenyl compounds (A1-31 and B1-17) were designed as programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) inhibitors. All compounds showed significant inhibitory activity with IC50 values ranging from 2.7 to 87.4 nM except compound A17, and compound B2 displayed the best activity. Further experiments showed that B2 bound to the PD-L1 protein without obvious toxicity in Lewis lung carcinoma (LLC) cells. Furthermore, B2 significantly promoted interferon-gamma secretion in a dose-dependent manner in vitro and in vivo. Especially, B2 exhibited potent in vivo anticancer efficacy in an LLC-bearing allograft mouse model at a low dose of 5 mg/kg, which was more active than BMS-1018 (tumor growth inhibition rate: 48.5% vs 17.8%). A panel of immunohistochemistry and flow cytometry assays demonstrated that B2 effectively counteracted PD-1-induced immunosuppression in the tumor microenvironment, thereby triggering antitumor immunity. These results indicate that B2 is a promising PD-1/PD-L1 inhibitor worthy of further development.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Desenho de Fármacos , Inibidores de Checkpoint Imunológico/síntese química , Nitrobenzenos/química , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Humanos , Inibidores de Checkpoint Imunológico/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Interferon gama/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Nitrobenzenos/metabolismo , Nitrobenzenos/farmacologia , Nitrobenzenos/uso terapêutico , Receptor de Morte Celular Programada 1/metabolismo , Relação Estrutura-Atividade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Stem Cell Reports ; 16(5): 1165-1181, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: covidwho-1225410

RESUMO

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71+ erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45+ CD71+ cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.


Assuntos
Imunidade Adaptativa/imunologia , COVID-19/patologia , Células Precursoras Eritroides/virologia , Eritropoese/fisiologia , Hemoglobinas/análise , Oxigênio/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , COVID-19/imunologia , Dexametasona/farmacologia , Células Precursoras Eritroides/imunologia , Feminino , Humanos , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Serina Endopeptidases/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Adulto Jovem
9.
J Med Chem ; 64(11): 7371-7389, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34029463

RESUMO

The previous virtual screening of ten million compounds yielded two novel nonlipopeptide-like chemotypes as TLR2 agonists. Herein, we present the chemical optimization of our initial hit, 1-phenyl-3-(thiophen-2-yl)urea, which resulted in the identification of SMU-C80 (EC50 = 31.02 ± 1.01 nM) as a TLR2-specific agonist with a 370-fold improvement in bioactivity. Mechanistic studies revealed that SMU-C80, through TLR1/2, recruits the adaptor protein MyD88 and triggers the NF-κB pathway to release cytokines such as TNF-α and IL-1ß from human, but not murine, cells. To the best of our knowledge, it is the first species-specific TLR1/2 agonist reported until now. Moreover, SMU-C80 increased the percentage of T, B, and NK cells ex vivo and activated the immune cells, which suppressed cancer cell growth in vitro. In summary, we obtained a highly efficient and specific human TLR1/2 agonist that acts through the MyD88 and NF-κB pathway, facilitating cytokine release and the simultaneous activation of immune cells that in turn affects the apoptosis of cancer cells.


Assuntos
Desenho de Fármacos , Tioureia/análogos & derivados , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Animais , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Imunoterapia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Neoplasias/terapia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tioureia/metabolismo , Tioureia/uso terapêutico , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/metabolismo
10.
Stem Cell Reports ; 16(5): 1165-1181, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33979601

RESUMO

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71+ erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45+ CD71+ cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.


Assuntos
Imunidade Adaptativa/imunologia , COVID-19/patologia , Células Precursoras Eritroides/virologia , Eritropoese/fisiologia , Hemoglobinas/análise , Oxigênio/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , COVID-19/imunologia , Dexametasona/farmacologia , Células Precursoras Eritroides/imunologia , Feminino , Humanos , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Serina Endopeptidases/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Adulto Jovem
11.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801585

RESUMO

The metastasis suppressor function of NM23 proteins is widely understood. Multiple enzymatic activities of NM23 proteins have also been identified. However, relatively less known interesting aspects are being revealed from recent developments that corroborate the telomeric interactions of NM23 proteins. Telomeres are known to regulate essential physiological events such as metastasis, ageing, and cellular differentiation via inter-connected signalling pathways. Here, we review the literature on the association of NM23 proteins with telomeres or telomere-related factors, and discuss the potential implications of emerging telomeric functions of NM23 proteins. Further understanding of these aspects might be instrumental in better understanding the metastasis suppressor functions of NM23 proteins.


Assuntos
Envelhecimento , Regulação Neoplásica da Expressão Gênica , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Telômero/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Proliferação de Células , Citoesqueleto/metabolismo , DNA/química , Quadruplex G , Humanos , Ativação Linfocitária , Mitocôndrias/metabolismo , Nucleosídeo Difosfato Quinase D/química , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Telômero/ultraestrutura , Fatores de Transcrição/metabolismo
12.
Front Immunol ; 12: 613591, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790891

RESUMO

T cells communicate with the environment via surface receptors. Cooperation of surface receptors regulates T-cell responses to diverse stimuli. Recently, finger-like membrane protrusions, microvilli, have been demonstrated to play a role in the organization of receptors and, hence, T-cell activation. However, little is known about the morphogenesis of dynamic microvilli, especially in the cells of immune system. In this review, I focus on the potential role of lipids and lipid domains in morphogenesis of microvilli. Discussed is the option that clustering of sphingolipids with phosphoinositides at the plasma membrane results in dimpling (curved) domains. Such domains can attract phosphoinositide-binding proteins and stimulate actin cytoskeleton reorganization. This process triggers cortical actin opening and bundling of actin fibres to support the growing of microvilli. Critical regulators of microvilli morphogenesis in T cells are unknown. At the end, I suggest several candidates with a potential to organize proteins and lipids in these structures.


Assuntos
Metabolismo dos Lipídeos , Microvilosidades/metabolismo , Linfócitos T/citologia , Linfócitos T/fisiologia , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Imunomodulação , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Microvilosidades/ultraestrutura , Morfogênese , Fosfatidilinositóis/metabolismo , Ligação Proteica , Transdução de Sinais , Esfingolipídeos/metabolismo , Linfócitos T/ultraestrutura
13.
FASEB J ; 35(5): e21577, 2021 05.
Artigo em Inglês | MEDLINE | ID: covidwho-1172658

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is an emerging respiratory pathogen that has rapidly spread in human populations. Severe forms of infection associate cytokine release syndrome and acute lung injury due to hyperinflammatory responses even though virus clearance is achieved. Key components of inflammation include immune cell recruitment in infected tissues, a step which is under the control of endothelial cells. Here, we review endothelial cell responses in inflammation and infection due to SARS-CoV-2 together with phenotypic and functional alterations of monocytes, T and B lymphocytes with which they interact. We surmise that endothelial cells function as an integrative and active platform for the various cells recruited, where fine tuning of immune responses takes place and which provides opportunities for therapeutic intervention.


Assuntos
Imunidade Adaptativa/imunologia , COVID-19/imunologia , COVID-19/patologia , Células Endoteliais/patologia , Células Mieloides/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Citocinas/imunologia , Humanos , Memória Imunológica , Células Mieloides/citologia , Linfócitos T/citologia , Linfócitos T/imunologia
14.
FASEB J ; 35(5): e21577, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33831263

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is an emerging respiratory pathogen that has rapidly spread in human populations. Severe forms of infection associate cytokine release syndrome and acute lung injury due to hyperinflammatory responses even though virus clearance is achieved. Key components of inflammation include immune cell recruitment in infected tissues, a step which is under the control of endothelial cells. Here, we review endothelial cell responses in inflammation and infection due to SARS-CoV-2 together with phenotypic and functional alterations of monocytes, T and B lymphocytes with which they interact. We surmise that endothelial cells function as an integrative and active platform for the various cells recruited, where fine tuning of immune responses takes place and which provides opportunities for therapeutic intervention.


Assuntos
Imunidade Adaptativa/imunologia , COVID-19/imunologia , COVID-19/patologia , Células Endoteliais/patologia , Células Mieloides/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Citocinas/imunologia , Humanos , Memória Imunológica , Células Mieloides/citologia , Linfócitos T/citologia , Linfócitos T/imunologia
15.
Nat Commun ; 12(1): 2437, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893286

RESUMO

CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR ( http://z.umn.edu/spliceR ) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.


Assuntos
Adenosina/metabolismo , Sistemas CRISPR-Cas , Biologia Computacional/métodos , Citidina/metabolismo , Edição de Genes/métodos , Adenosina/química , Sequência de Bases , Células Cultivadas , Citidina/química , Humanos , Internet , Células K562 , Reprodutibilidade dos Testes , Linfócitos T/citologia , Linfócitos T/metabolismo
17.
Front Immunol ; 12: 604066, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679741

RESUMO

Expansion protocols for human T lymphocytes using magnetic beads, which serve as artificial antigen presenting cells (aAPCs), is well-studied. Yet, the efficacy of magnetic beads for propagation and functionality of peripheral blood lymphocytes (PBLs) isolated from companion dogs still remains limited. Domestic dog models are important in immuno-oncology field. Thus, we built the platform for induction of canine PBLs function, proliferation and biological activity using nano-sized magnetic beads (termed as MicroBeads) coated with anti-canine CD3 and CD28 antibodies. Herein we reveal that activation of canine PBLs via MicroBeads induces a range of genes involved in immediate-early response to T cell activation in dogs. Furthermore, canine T lymphocytes are effectively activated by MicroBeads, as measured by cluster formation and induction of activation marker CD25 on canine T cells as quickly as 24 h post stimulation. Similar to human T cells, canine PBLs require lower activation signal strength for efficient proliferation and expansion, as revealed by titration studies using a range of MicroBeads in the culture. Additionally, the impact of temperature was assessed in multiple stimulation settings, showing that both 37°C and 38.5°C are optimal for the expansion of canine T cells. In contrast to stimulation using plant mitogen Concanavalin A (ConA), MicroBead-based activation did not increase activation-induced cell death. In turn, MicroBeads supported the propagation of T cells with an effector memory phenotype that secreted substantial IL-2 and IFN-γ. Thus, MicroBeads represent an accessible and affordable tool for conducting immunological studies on domestic dog models. Similarities in inducing intracellular signaling pathways further underscore the importance of this model in comparative medicine. Presented herein MicroBead-based expansion platforms for canine PBLs may benefit adoptive immunotherapy in dogs and facilitate the design of next-generation clinical trials in humans.


Assuntos
Proliferação de Células , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Cães , Linfócitos T/citologia
18.
Biochem Biophys Res Commun ; 550: 70-76, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33689882

RESUMO

T cells secrete several inflammatory cytokines that play a critical role in the progression of atherosclerosis. Although green tea epigallocatechin-3-gallate (EGCG) exerts anti-inflammatory and anti-atherosclerotic effects in animals, few studies have identified the mechanism underlying these effects in human primary T cells. This study investigated the pathway involved in EGCG modulation of cytokine secretion in activated human primary T cells. We pre-treated human primary T cells with EGCG (0.1, 1, 5, 10, and 20 µM) for 4 h and incubated them with or without phorbol 12-myristate 13-acetate and ionomycin (P/I) for 20 h. The cytokine production, activator protein (AP)-1 binding activity, and level of mitogen-activated protein kinase (MAPK) were assessed using enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blotting, respectively. At 10 and 20 µM, EGCG decreased interleukin (IL)-2 levels by 26.0% and 38.8%, IL-4 levels by 41.5% and 55.9%, INF-γ levels by 31.3% and 34.7%, and tumor-necrosis factor (TNF)-α levels by 23.0% and 37.6%, respectively. In addition, the level of phosphorylated c-Jun N-terminal (p-JNK) and extracellular signal-regulated kinase (p-ERK) was decreased, but not the level of p-p38 MAPK. EGCG did not alter any of the total protein amounts, suggesting a selective effect on specific types of MAPKs in stimulated human T cells. EGCG tended to inactivate AP-1 DNA-binding activity. The P/I-induced production of IL-2, IL-4, INF-γ, and TNF-α by human T cells was suppressed by AP-1 inhibitor in a concentration-dependent manner. In conclusion, EGCG suppressed cytokine secretion in activated human primary T cells, and this effect was likely mediated by AP-1 inactivation through the ERK and JNK, but not p38 MAPK, pathways. These results may be related to the mechanisms through which EGCG inhibits immune- or inflammation-related atherogenesis.


Assuntos
Catequina/análogos & derivados , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/farmacologia , Catequina/imunologia , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Ativação Linfocitária/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/metabolismo , Fator de Transcrição AP-1/metabolismo
19.
Cell Prolif ; 54(4): e13022, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33686740

RESUMO

OBJECTIVES: This study aimed to investigate the protective effect of SCARF1 on acute rejection (AR), phagocytic clearance of Kupffer cells (KCs), M2 polarization and the exact mechanism underlying these processes. METHODS: AAV was transfected into the portal vein of rats, and AR and immune tolerance (IT) models of liver transplantation were established. Liver tissue and blood samples were collected. The level of SCARF1 was detected via WB and immunohistochemical staining. Pathological changes in liver tissue were detected using HE staining. Apoptotic cells were detected using TUNEL staining. KC polarization was assessed via immunohistochemical staining. Primary KCs were isolated and co-cultured with apoptotic T lymphocytes. Phagocytosis of apoptotic cells and polarization of KCs were both detected using immunofluorescence. Calcium concentration was determined using immunofluorescence and a fluorescence microplate reader. The levels of PI3K, p-AKT and P-STAT3 were assessed via WB and immunofluorescence. RESULTS: Compared to the IT group, the level of SCARF1 was significantly decreased in the AR group. Overexpression of SCARF1 in KCs improved AR and liver function markers. Enhanced phagocytosis mediated by SCARF1 is beneficial for improving the apoptotic clearance of AR and promoting M2 polarization of KCs. SCARF1-mediated enhancement of phagocytosis promotes increased calcium concentration in KCs, thus further activating the PI3K-AKT-STAT3 signalling pathway. CONCLUSIONS: SCARF1 promotes the M2 polarization of KCs by promoting phagocytosis through the calcium-dependent PI3K-AKT-STAT3 signalling pathway.


Assuntos
Cálcio/metabolismo , Transplante de Fígado , Receptores Depuradores Classe F/metabolismo , Transdução de Sinais , Animais , Apoptose , Polaridade Celular , Células Cultivadas , Técnicas de Cocultura , Macrófagos do Fígado/citologia , Macrófagos do Fígado/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Fagocitose , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos Lew , Fator de Transcrição STAT3/metabolismo , Receptores Depuradores Classe F/genética , Linfócitos T/citologia , Linfócitos T/metabolismo
20.
Nat Commun ; 12(1): 1112, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: covidwho-1091491

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide health threat. In a prospective multicentric study, we identify IL-3 as an independent prognostic marker for the outcome during SARS-CoV-2 infections. Specifically, low plasma IL-3 levels is associated with increased severity, viral load, and mortality during SARS-CoV-2 infections. Patients with severe COVID-19 exhibit also reduced circulating plasmacytoid dendritic cells (pDCs) and low plasma IFNα and IFNλ levels when compared to non-severe COVID-19 patients. In a mouse model of pulmonary HSV-1 infection, treatment with recombinant IL-3 reduces viral load and mortality. Mechanistically, IL-3 increases innate antiviral immunity by promoting the recruitment of circulating pDCs into the airways by stimulating CXCL12 secretion from pulmonary CD123+ epithelial cells, both, in mice and in COVID-19 negative patients exhibiting pulmonary diseases. This study identifies IL-3 as a predictive disease marker for SARS-CoV-2 infections and as a potential therapeutic target for pulmunory viral infections.


Assuntos
COVID-19/diagnóstico , Interleucina-3/sangue , Animais , COVID-19/mortalidade , Quimiocina CXCL12/imunologia , Células Dendríticas/citologia , Modelos Animais de Doenças , Feminino , Alemanha , Humanos , Imunidade Inata , Interferons/sangue , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estudos Prospectivos , Índice de Gravidade de Doença , Linfócitos T/citologia , Carga Viral
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