RESUMO
The development and function of natural killer (NK) cells is regulated by the interaction of inhibitory receptors of the Ly49 family with distinct peptide-laden major histocompatibility complex (MHC) class I molecules, although whether the Ly49 family is able bind to other MHC class I-like molecules is unclear. Here we found that the prototypic inhibitory receptor Ly49A bound the highly conserved nonclassical MHC class I molecule H2-M3 with an affinity similar to its affinity for H-2D(d). The specific recognition of H2-M3 by Ly49A regulated the 'licensing' of NK cells and mediated 'missing-self' recognition of H2-M3-deficient bone marrow. Host peptide-H2-M3 was required for optimal NK cell activity against experimental metastases and carcinogenesis. Thus, nonclassical MHC class I molecules can act as cognate ligands for Ly49 molecules. Our results provide insight into the various mechanisms that lead to NK cell tolerance.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Animais , Antígenos de Histocompatibilidade Classe I/genética , Tolerância Imunológica , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Cytotoxic lymphocytes rapidly respond and destroy both malignant cells and cells infected with intracellular pathogens. One mechanism, known as granule exocytosis, employs the secretory granules of these lymphocytes. These include the pore-forming protein perforin (pfp) and a family of serine proteases known as granzymes that cleave and activate effector molecules within the target cell. Over the past two decades, the study of granzymes has largely focused on the ability of these serine proteases to induce cell death. More recently, sophisticated mouse models of disease coupled with gene-targeted mice have allowed investigators to ask why granzyme subfamilies are encoded on different chromosomal loci and what broader role these enzymes might play in inflammation and immune response. Here, we provide a brief overview of the granzyme superfamily, their relationship to pfp, and their reported functions in apoptosis. This overview is followed by a comprehensive analysis of the less characterized and developing field regarding the non-apoptotic functions of granzymes.
Assuntos
Apoptose , Citotoxicidade Imunológica , Granzimas/metabolismo , Inflamação/enzimologia , Linfócitos T Citotóxicos/enzimologia , Animais , Apoptose/genética , Citotoxicidade Imunológica/genética , Modelos Animais de Doenças , Granzimas/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Perforina/metabolismo , Vesículas Secretórias/enzimologia , Vesículas Secretórias/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.
RESUMO
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies. However, the underlying cause of disease appears to relate to defects in T cell tolerance or T cell help to B cells. Transgenic (Tg) mice overexpressing the cytokine B cell-activating factor of the tumor necrosis factor family (BAFF) develop an autoimmune disorder similar to SLE and show impaired B cell tolerance and altered T cell differentiation. We generated BAFF Tg mice that were completely deficient in T cells, and, surprisingly, these mice developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did, however, require B cell-intrinsic signals through the Toll-like receptor (TLR)-associated signaling adaptor MyD88, which controlled the production of proinflammatory autoantibody isotypes. TLR7/9 activation strongly up-regulated expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which is a receptor for BAFF involved in B cell responses to T cell-independent antigens. Moreover, BAFF enhanced TLR7/9 expression on B cells and TLR-mediated production of autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, but requires TLR signaling and is independent of T cell help. It is possible that SLE patients with elevated levels of BAFF show a similar basis for disease.
Assuntos
Fator Ativador de Células B/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , Animais , Autoanticorpos/química , Fator Ativador de Células B/metabolismo , Diferenciação Celular , Separação Celular , Regulação da Expressão Gênica , Imunoglobulina A/metabolismo , Imunoglobulina M/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Fator 88 de Diferenciação Mieloide/metabolismo , Linfócitos T/metabolismoRESUMO
Lymphocyte perforin and serine protease granzymes are well-recognized extrinsic mediators of apoptosis. We now demonstrate that cytotoxic lymphocyte granule components profoundly augment the myeloid cell inflammatory cytokine cascade in response to TLR4 ligation. Whereas caspase-1-deficient mice were completely resistant to LPS, reduced serum cytokine production and resistance to lethal endotoxicosis were also obtained with perforin-deficient mice, indicating a role for granzymes. Consistently, a lack of granzyme M (GrzM) resulted in reduced serum IL-1alpha, IL-1beta, TNF, and IFN-gamma levels and significantly reduced susceptibility to lethal endotoxicosis. These altered responses were also observed in granzyme A-deficient but not granzyme B-deficient mice. A role for APC-NK cell cross-talk in the inflammatory cascade was highlighted, as GrzM was exclusively expressed by NK cells and resistance to LPS was also observed on a RAG-1/GrzM-double deficient background. Collectively, the data suggest that NK cell GrzM augments the inflammatory cascade downstream of LPS-TLR4 signaling, which ultimately results in lethal endotoxicosis. Most importantly, these data demonstrate that granzymes should no longer be considered solely as mediators of apoptosis, but additionally as potential key regulators of inflammation.
Assuntos
Granzimas/fisiologia , Mediadores da Inflamação/toxicidade , Choque Séptico/enzimologia , Choque Séptico/patologia , Receptor 4 Toll-Like/fisiologia , Animais , Granzimas/deficiência , Granzimas/genética , Imunidade Inata/imunologia , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Ligantes , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/enzimologia , Células Mieloides/imunologia , Células Mieloides/patologia , Choque Séptico/mortalidade , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/metabolismoRESUMO
Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8(+) T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.
Assuntos
Apresentação de Antígeno/imunologia , Divisão Celular/imunologia , Sequência Conservada/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Adesão Celular/imunologia , Polaridade Celular/imunologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subpopulações de Linfócitos T/metabolismoRESUMO
The mechanism of action of eprenetapopt (APR-246, PRIMA-1MET) as an anticancer agent remains unresolved, although the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis (SLC7A11, SHMT2, and MTHFD1L), as well as the enzymes required to synthesize glutathione (GCLC and GCLM), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limiting the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.
RESUMO
Toll-like receptor-4-lipopolysaccharide (LPS)-mediated inflammation is used to delineate signals involved in cross-talk between antigen-presenting cells (APCs) and lymphocytes such as natural killer (NK) cells. Following APC stimulation and cytokine release, NK cells produce interferon (IFN)-γ. High levels of LPS induce endotoxicosis, a systemic inflammatory disease in which IFN-γ causes significant morbidity and mortality. Several studies have highlighted the role of interleukin (IL)-18, IL-1ß, IL-17A and IFN-γ in the development of endotoxicosis, but whether these cytokines interact with each other is yet to be determined. Our data demonstrate that IL-18 and IL-17A have important roles in NK cell IFN-γ production during endotoxicosis. Importantly, we provide the first evidence that IL-18 also has a role in IL-17A production by T-cell receptor (TCR)-δ cells. Furthermore, we demonstrate that IL-18-deficient mice have a defect in γδ T-cell homeostasis and IL-1ß production, both of which can contribute to the development of disease through induction of IL-17A. These results reveal novel requirements for IL-18 in innate immune cell homeostasis and activation, demonstrating that the role of IL-18 in innate immunity occurs at a level other than activation.
Assuntos
Interleucina-18/fisiologia , Lipopolissacarídeos/toxicidade , Animais , Células Cultivadas , Homeostase/genética , Homeostase/imunologia , Inflamação/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-18/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologiaRESUMO
NK cells have been proposed to be an initial source of IFN-gamma that supports either Th1 or CTL priming. Although NK cells reside in naive lymph nodes (LN) at a very low frequency, they can be recruited into LN draining sites of infection, inflammation, or immunization where they potentially influence adaptive immunity. In this study, we report that mature CD27(high) NK cells are predominantly recruited into the draining LN following dendritic cell (DC) challenge. Importantly, the recruitment of the CD27(high) NK cell subset in the draining LN was dependent on host IFN-gamma and the activation status of NK cells. Endogenous epidermal DC migration induced by hapten challenge also triggers NK cell recruitment to the draining LN in an IFN-gamma-dependent mechanism. Thus, our results identify that CD27(high) NK cells are the dominant population recruited to the draining LN and NK cell recruitment requires endogenous IFN-gamma in coordinating with DC migration.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Movimento Celular/genética , Células Dendríticas/citologia , Interferon gama/genética , Linfonodos/citologia , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genéticaRESUMO
We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.
Assuntos
Granzimas/química , Granzimas/metabolismo , Muromegalovirus/imunologia , Peptídeos/metabolismo , Animais , Apoptose , Caspases/metabolismo , Morte Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Granzimas/genética , Infecções por Herpesviridae/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Peptídeos/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Especificidade por Substrato , Linfócitos T Citotóxicos/imunologia , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Proteína bcl-X/metabolismoRESUMO
The acquisition of inhibitory MHC-specific receptors occurs during NK cell differentiation and has been considered important in regulating NK cell responsiveness. NK cell differentiation has been studied on the basis of cell surface phenotype, function, and proliferative capacity. Together with phenotypically immature Mac-1lo NK cells, the mature Mac-1hi NK cell pool can be dissected further into two functionally distinct CD27hi and CD27lo subsets. Two major inhibitory receptors, CD94/NKG2A and Ly-49, are expressed on mouse NK cells. The acquisition of the CD94/NKG2A receptor seems to be an early event, whereas Ly-49 receptor expression is considered a relatively late event during NK cell ontogeny. In this study, we demonstrated a distinct NK cell inhibitory receptor repertoire formation within mature NK cell populations as defined by Mac-1 and CD27. By analyzing mice deficient in MHC class I expression or NKG2D ligand transgenic mice, we have shown that the inhibitory receptor repertoire can be modulated according to the differentiation/maturation status of NK cells, and the receptor acquisition is imprinted at an early stage of NK cell development by MHC class I interactions.