RESUMO
Infection of mice by mouse cytomegalovirus (MCMV) triggers activation and expansion of Ly49H+ natural killer (NK) cells, which are virus specific and considered to be "adaptive" or "memory" NK cells. Here, we find that signaling lymphocytic activation molecule family receptors (SFRs), a group of hematopoietic cell-restricted receptors, are essential for the expansion of Ly49H+ NK cells after MCMV infection. This activity is largely mediated by CD48, an SFR broadly expressed on NK cells and displaying augmented expression after MCMV infection. It is also dependent on the CD48 counter-receptor, 2B4, expressed on host macrophages. The 2B4-CD48 axis promotes expansion of Ly49H+ NK cells by repressing their phagocytosis by virus-activated macrophages through inhibition of the pro-phagocytic integrin lymphocyte function-associated antigen-1 (LFA-1) on macrophages. These data identify key roles of macrophages and the 2B4-CD48 pathway in controlling the expansion of adaptive NK cells following MCMV infection. Stimulation of the 2B4-CD48 axis may be helpful in enhancing adaptive NK cell responses for therapeutic purposes.
Assuntos
Infecções por Citomegalovirus , Receptores Imunológicos , Animais , Camundongos , Receptores Imunológicos/metabolismo , Antígeno CD48/metabolismo , Antígenos CD/metabolismo , Ativação Linfocitária , Células Matadoras Naturais , Receptores de Superfície Celular/metabolismo , Proteínas de Transporte/metabolismo , Macrófagos/metabolismo , FagocitoseRESUMO
BACKGROUND: SARS-CoV-2 remains a world-wide health issue. SARS-CoV-2-specific immunity is induced upon both infection and vaccination. However, defining the long-term immune trajectory, especially after infection, is limited. In this study, we aimed to further the understanding of long-term SARS-CoV-2-specific immune response after infection. RESULTS: We conducted a longitudinal cohort study among 93 SARS-CoV-2 recovered individuals. Immune responses were continuously monitored for up to 20 months after infection. The humoral responses were quantified by Spike- and Nucleocapsid-specific IgG levels. T cell responses to Spike- and non-Spike epitopes were examined using both intercellular cytokine staining (ICS) assay and Activation-Induced marker (AIM) assay with quantification of antigen-specific IFNγ production. During the 20 months follow-up period, Nucleocapsid-specific antibody levels and non-Spike-specific CD4 + and CD8 + T cell frequencies decreased in the blood. However, a majority of participants maintained a durable immune responses 20 months after infection: 59% of the participants were seropositive for Nucleocapsid-specific IgG, and more than 70% had persisting non-Spike-specific T cells. The Spike-specific response initially decreased but as participants were vaccinated against COVID-19, Spike-specific IgG levels and T cell frequencies were boosted reaching similar or higher levels compared to 1 month post-infection. The trajectory of infection-induced SARS-CoV-2-specific immunity decreases, but for the majority of participants it persists beyond 20 months. The T cell response displays a greater durability. Vaccination boosts Spike-specific immune responses to similar or higher levels as seen after primary infection. CONCLUSIONS: For most participants, the response persists 20 months after infection, and the cellular response appears to be more long-lived compared to the circulating antibody levels. Vaccination boosts the S-specific response but does not affect the non-S-specific response. Together, these findings support the understanding of immune contraction, and with studies showing the immune levels required for protection, adds to the knowledge of durability of protection against future SARS-CoV-2.
Assuntos
COVID-19 , Humanos , Estudos Longitudinais , SARS-CoV-2 , Imunidade Celular , Imunoglobulina G , Anticorpos Antivirais , Imunidade Humoral , VacinaçãoRESUMO
Cancer cells elude anti-tumour immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors. Phagocytosis by macrophages plays a critical role in cancer control. Therapeutic blockade of signal regulatory protein (SIRP)-α, an inhibitory receptor on macrophages, or of its ligand CD47 expressed on tumour cells, improves tumour cell elimination in vitro and in vivo, suggesting that blockade of the SIRPα-CD47 checkpoint could be useful in treating human cancer. However, the pro-phagocytic receptor(s) responsible for tumour cell phagocytosis is(are) largely unknown. Here we find that macrophages are much more efficient at phagocytosis of haematopoietic tumour cells, compared with non-haematopoietic tumour cells, in response to SIRPα-CD47 blockade. Using a mouse lacking the signalling lymphocytic activation molecule (SLAM) family of homotypic haematopoietic cell-specific receptors, we determined that phagocytosis of haematopoietic tumour cells during SIRPα-CD47 blockade was strictly dependent on SLAM family receptors in vitro and in vivo. In both mouse and human cells, this function required a single SLAM family member, SLAMF7 (also known as CRACC, CS1, CD319), expressed on macrophages and tumour cell targets. In contrast to most SLAM receptor functions, SLAMF7-mediated phagocytosis was independent of signalling lymphocyte activation molecule-associated protein (SAP) adaptors. Instead, it depended on the ability of SLAMF7 to interact with integrin Mac-1 (refs 18, 19, 20) and utilize signals involving immunoreceptor tyrosine-based activation motifs. These findings elucidate the mechanism by which macrophages engulf and destroy haematopoietic tumour cells. They also reveal a novel SAP adaptor-independent function for a SLAM receptor. Lastly, they suggest that patients with tumours expressing SLAMF7 are more likely to respond to SIRPα-CD47 blockade therapy.
Assuntos
Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Antígeno de Macrófago 1/metabolismo , Macrófagos/imunologia , Fagocitose/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Actinas/metabolismo , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Feminino , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/deficiênciaRESUMO
Signaling lymphocytic activation molecule (SLAM) family receptors (SFRs) can mediate either activating or inhibitory effects during natural killer cell (NK cell) activation. In this study, we addressed the global role, regulation, and mechanism of action of the SLAM family in NK cells by analyzing a mouse lacking the entire â¼400-kilobase Slam locus, which encodes all six SFRs and CD48, the ligand of SFR 2B4. This mouse displayed enhanced NK cell activation responses toward hematopoietic target cells. Analyses of mice lacking individual SFRs showed that the inhibitory function of the Slam locus was due solely to 2B4 and was not influenced positively or negatively by other SFRs. Differences in NK cell responses between recognition of targets expressing or lacking ligands for SFRs were enhanced by IL-12 but suppressed by type I interferon. Cytokines also changed the levels of SLAM-associated protein adaptors, which prevent the inhibitory function of SFRs. The enhanced activation responses of SFR-deficient NK cells were dependent on integrin LFA-1 but not on DNAM-1 or NKG2D. SFR-mediated inhibition prevented the generation of activated forms of LFA-1. Hence, the Slam locus has an overall inhibitory role during NK cell activation that is solely dependent on 2B4. This effect is influenced by cytokines and leads to suppression of LFA-1 activity.
Assuntos
Citocinas/metabolismo , Deleção de Genes , Loci Gênicos , Células Matadoras Naturais/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Animais , Citotoxicidade Imunológica , Hematopoese , Humanos , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica , Camundongos Endogâmicos C57BL , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Linfócitos T/metabolismoRESUMO
Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.
Assuntos
Antígenos CD/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Matadoras Naturais/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Receptores de Superfície Celular/imunologia , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , Imunidade Inata , Ativação Linfocitária , Melanoma Experimental , Camundongos , Transdução de Sinais , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
Signaling lymphocytic activation molecule F7 (SLAMF7) is a receptor present on immune cells, including natural killer (NK) cells. It is also expressed on multiple myeloma (MM) cells. This led to development of an anti-SLAMF7 antibody, elotuzumab, showing efficacy against MM. SLAMF7 mediates activating or inhibitory effects in NK cells, depending on whether cells express or do not express the adaptor EAT-2. Since MM cells lack EAT-2, we elucidated the inhibitory effectors of SLAMF7 in EAT-2-negative NK cells and tested whether these effectors were triggered in MM cells. SLAMF7-mediated inhibition in NK cells lacking EAT-2 was mediated by SH2 domain-containing inositol phosphatase 1 (SHIP-1), which was recruited via tyrosine 261 of SLAMF7. Coupling of SLAMF7 to SHIP-1 required Src kinases, which phosphorylated SLAMF7. Although MM cells lack EAT-2, elotuzumab did not induce inhibitory signals in these cells. This was at least partly due to a lack of CD45, a phosphatase required for Src kinase activation. A defect in SLAMF7 function was also observed in CD45-deficient NK cells. Hence, SLAMF7-triggered inhibition is mediated by a mechanism involving Src kinases, CD45, and SHIP-1 that is defective in MM cells. This defect might explain why elotuzumab eliminates MM cells by an indirect mechanism involving the activation of NK cells.
Assuntos
Células Matadoras Naturais/citologia , Antígenos Comuns de Leucócito/metabolismo , Mieloma Múltiplo/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Receptores Imunológicos/metabolismo , Quinases da Família src/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Células COS , Chlorocebus aethiops , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Inositol Polifosfato 5-Fosfatases , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosforilação , Estrutura Terciária de Proteína , Interferência de RNA , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária , Tirosina/químicaRESUMO
Human Galectin-3 (Gal-3), a ß-galactoside-binding protein expressed by tumor cells, has been reported to act as an immune regulator in antitumor T cells. However, its effect on natural killer (NK) cells is elusive. Using a recombinant human NK cell-activating receptor, NKp30 fusion protein (NKp30-Fc), we found that soluble NKp30-Fc could immunoprecipitate Galectin-3. The direct interaction between NKp30 and Galectin-3 was further confirmed using surface plasmon resonance experiments. Because Galectin-3 was mainly released from tumor cells in a soluble form in our study, the binding assay was performed to show that soluble Galectin-3 specifically bound to NK cells and NKp30 on the surface of the NK cells. Functionally, when soluble Galectin-3 was added to the NK-tumor cell coculture system, the NKp30-mediated, but not NKG2D-mediated, cytolysis and CD107a expression in the NK cells were inhibited, and these phenotypes could be restored by preincubation of soluble Galectin-3 with NKp30-Fc fusion protein or the addition of anti-Gal-3 antibody alone. Moreover, genetic down-regulation of Galectin-3 (shGal-3) resulted in tumor cells being more sensitive to NK cell lysis, and, reversely, Galectin-3-overexpressing HeLa cells (exGal-3) became less sensitive to NK cell killing. The results of these in vitro experiments were supported by studies in shGal-3-HeLa or exGal-3-HeLa xenograft non-obese diabetic/severe combined immunodeficiency mice after NK cell adoptive immunotherapy, indicating that Galectin-3 strongly antagonizes human NK cell attack against tumors in vivo. These findings indicate that Galectin-3 may function as an immune regulator to inhibit NK cell function against tumors, therefore providing a new therapeutic target for tumor treatment.
Assuntos
Galectina 3/imunologia , Imunidade Celular , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Evasão Tumoral , Animais , Proteínas Sanguíneas , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Galectina 3/genética , Galectinas , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Células HeLa , Xenoenxertos , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cálcio/metabolismo , Grânulos Citoplasmáticos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Fosfolipase C gama/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática , Exocitose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Dados de Sequência Molecular , Fosfolipase C gama/química , Fosforilação , Estrutura Terciária de Proteína , Transdução de Sinais , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Relação Estrutura-Atividade , Fatores de Transcrição/química , Tirosina/metabolismoRESUMO
Herpes simplex encephalitis (HSE) is a lethal neurological disease resulting from infection with Herpes Simplex Virus 1 (HSV-1). Loss-of-function mutations in the UNC93B1, TLR3, TRIF, TRAF3, and TBK1 genes have been associated with a human genetic predisposition to HSE, demonstrating the UNC93B-TLR3-type I IFN pathway as critical in protective immunity to HSV-1. However, the TLR3, UNC93B1, and TRIF mutations exhibit incomplete penetrance and represent only a minority of HSE cases, perhaps reflecting the effects of additional host genetic factors. In order to identify new host genes, proteins and signaling pathways involved in HSV-1 and HSE susceptibility, we have implemented the first genome-wide mutagenesis screen in an in vivo HSV-1 infectious model. One pedigree (named P43) segregated a susceptible trait with a fully penetrant phenotype. Genetic mapping and whole exome sequencing led to the identification of the causative nonsense mutation L3X in the Receptor-type tyrosine-protein phosphatase C gene (Ptprc(L3X)), which encodes for the tyrosine phosphatase CD45. Expression of MCP1, IL-6, MMP3, MMP8, and the ICP4 viral gene were significantly increased in the brain stems of infected Ptprc(L3X) mice accounting for hyper-inflammation and pathological damages caused by viral replication. Ptprc(L3X) mutation drastically affects the early stages of thymocytes development but also the final stage of B cell maturation. Transfer of total splenocytes from heterozygous littermates into Ptprc(L3X) mice resulted in a complete HSV-1 protective effect. Furthermore, T cells were the only cell population to fully restore resistance to HSV-1 in the mutants, an effect that required both the CD4⺠and CD8⺠T cells and could be attributed to function of CD4⺠T helper 1 (Th1) cells in CD8⺠T cell recruitment to the site of infection. Altogether, these results revealed the CD45-mediated T cell function as potentially critical for infection and viral spread to the brain, and also for subsequent HSE development.
Assuntos
Códon sem Sentido , Encefalite por Herpes Simples/genética , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Imunidade Celular , Antígenos Comuns de Leucócito/metabolismo , Células Th1/imunologia , Animais , Tronco Encefálico/imunologia , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Tronco Encefálico/virologia , Células Cultivadas , Cruzamentos Genéticos , Suscetibilidade a Doenças , Encefalite por Herpes Simples/etiologia , Feminino , Estudo de Associação Genômica Ampla , Herpes Simples/patologia , Herpes Simples/fisiopatologia , Herpes Simples/virologia , Antígenos Comuns de Leucócito/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutagênese , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Análise de Sobrevida , Células Th1/metabolismo , Células Th1/patologia , Células Th1/virologiaRESUMO
Signaling lymphocytic activation molecule (SLAM) family receptors have been implicated in normal immunity, immunodeficiencies and autoimmunity. CS1 (also known as CRACC, CD319 and SLAMF7) is a member of the SLAM family expressed on several normal hematopoietic cell types. It is also highly and nearly universally expressed on multiple myeloma (MM) cells. This review focuses on the biology of CS1, both in normal hematopoietic cells and in MM cells. It also discusses the preclinical and clinical data on the use of a humanized anti-CS1 monoclonal antibody, elotuzumab, for the treatment of MM. Based on current knowledge, CS1 is a compelling new target for the treatment of MM.