RESUMO
NK cell development and homeostasis require IL-15 produced by both hematopoietic and parenchymal cells. Certain hematopoietic IL-15 sources, such as macrophages and dendritic cells, are known, whereas the source of parenchymal IL-15 remains elusive. Using two types of adipocyte-specific Il15(-/-) mice, we identified adipocytes as a parenchymal IL-15 source that supported NK cell development nonredundantly. Both adipocyte-specific Il15(-/-) mice showed reduced IL-15 production specifically in the adipose tissue but impaired NK cell development in the spleen and liver in addition to the adipose tissue. We also found that the adipose tissue harbored NK progenitors as other niches (e.g. spleen) for NK cell development, and that NK cells derived from transplanted adipose tissue populated the recipient's spleen and liver. These findings suggest that adipocyte IL-15 contributes to systemic NK cell development by supporting NK cell development in the adipose tissue, which serves as a source of NK cells for other organs.
Assuntos
Adipócitos/citologia , Diferenciação Celular/imunologia , Interleucina-15/imunologia , Células Matadoras Naturais/citologia , Adipócitos/imunologia , Adipócitos/transplante , Tecido Adiposo/imunologia , Transferência Adotiva , Animais , Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Homeostase/imunologia , Interleucina-15/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Lectinas Tipo C , Fígado/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , RNA Mensageiro/biossíntese , Receptores Imunológicos/biossíntese , Baço/citologiaRESUMO
Because of increasing interest in the removal of immunosuppressive pathways in cancer, the combination of IL-2 with Abs to neutralize TGF-ß, a potent immunosuppressive cytokine, was assessed. Combination immunotherapy resulted in significantly greater antitumor effects. These were correlated with significant increases in the numbers and functionality of NK cells, NK cell progenitors, and activated CD8 T cells, resulting in the observed antitumor effects. Combination immunotherapy also was accompanied by lesser toxicities than was IL-2 therapy alone. Additionally, we observed a dual competition between NK cells and activated CD8 T cells such that, after immunotherapy, the depletion of either effector population resulted in the increased total expansion of the other population and compensatory antitumor effects. This study demonstrates the efficacy of this combination immunotherapeutic regimen as a promising cancer therapy and illustrates the existence of potent competitive regulatory pathways between NK cells and CD8 T cells in response to systemic activation.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Interleucina-2/uso terapêutico , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Proteína Ligante Fas/imunologia , Feminino , Imunoterapia/métodos , Células Matadoras Naturais/transplante , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília C de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Linfócitos T Reguladores/imunologia , Receptor fas/imunologiaRESUMO
Natural killer (NK) cells express inhibitory receptors with varied binding affinities to specific major histocompatibility complex class I (MHC-I) haplotypes. NK cells can be classified as licensed or unlicensed based on their ability or inability to bind MHC-I, respectively. The role of donor vs host MHC on their development after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not known. Following reciprocal MHC-disparate allogeneic transplants and during de novo NK-cell recovery, depletion of the licensed and not unlicensed population of NK cells as determined by the licensing patterns of donor MHC-I haplotypes, resulted in significantly increased susceptibility to murine cytomegalovirus (MCMV) infection. A corresponding expansion of the licensed Ly49H(+) NK cells occurred with greater interferon γ production by these cells than unlicensed NK cells in the context of donor MHC-I. Thus, NK licensing behavior to MCMV corresponds to the donor, and not recipient, MHC haplotype after allo-HSCT in mice.
Assuntos
Infecções por Citomegalovirus/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Animais , Células da Medula Óssea/citologia , Feminino , Haplótipos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Transplante HomólogoRESUMO
The dendritic cell vaccine DC-Ad-GM·CAIX is an active, specific immunotherapy with the potential of providing a safe and effective therapy against renal cell carcinoma (RCC). Using immunocompetent Balb/c mouse models we tested the efficacy and mechanism of the vaccine to prevent and treat the growth of a syngeneic RCC (RENCA) engineered to overexpress the human TAA carbonic anhydrase IX (NPR-IX). In a prevention model, NPR-IX tumor development was specifically and significantly delayed by 13 days in DC-Ad-GM·CAIX-treated mice (P < 0.001), tumor volumes were 79% smaller (day 24, P < 0.007), and body weight was maintained at study termination compared with the controls. Six of these mice remained tumor-free for > 1 year. In a treatment model, NPR-IX tumors remained smaller in DC-Ad-GM·CAIX-treated mice for 8 days (P < 0.002), achieving a 60% growth inhibition at termination. No vaccine-related organ toxicity was observed in either model. The critical mechanistic parameter separating responsive from nonresponsive tumors was hCAIX protein expression, demonstrated by aggressive growth of tumors that did not express hCAIX protein and in sham-treated mice (DC-Ad-Null). No murine serum anti-hCAIX antibodies were detected. Moreover, altered mechanisms of immunoediting as a means for immune evasion were suggested by differential gene expression (Ccl1, Hmgb1, Fgl2, Cd209a, and Klra2) and therapy evasion miRNAs (miR-1186, miR-98, miR-5097, miR-1942, and miR-708) in tumors that evaded DC-Ad-GM·CAIX immunotherapy. This is the first study in immunocompetent mice that provides a proof of concept for the specificity, efficacy, safety, and activity of the DC-Ad-GM·CAIX immunotherapy, forming the basis for a first-in-human phase I trial in RCC patients.
Assuntos
Vacinas Anticâncer/uso terapêutico , Anidrases Carbônicas/imunologia , Carcinoma de Células Renais/prevenção & controle , Carcinoma de Células Renais/terapia , Células Dendríticas/imunologia , Imunoterapia Adotiva , Neoplasias Renais/terapia , Animais , Anticorpos/sangue , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Anidrase Carbônica IX , Anidrases Carbônicas/biossíntese , Carcinoma de Células Renais/imunologia , Moléculas de Adesão Celular/biossíntese , Linhagem Celular Tumoral , Quimiocina CCL1/biossíntese , Modelos Animais de Doenças , Feminino , Fibrinogênio/biossíntese , Expressão Gênica , Neoplasias Renais/imunologia , Neoplasias Renais/prevenção & controle , Lectinas Tipo C/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Receptores de Superfície Celular/biossínteseRESUMO
NK cell receptors (NKRs) such as NK1.1, NKG2D, and Ly49s are expressed on subsets of CD1d-independent memory phenotype CD8(+) and CD4(-)CD8(-) T cells. However, the mechanism for the generation and functions of these NKR(+) T cells remained elusive. In this study, we found that CD1d-independent Ly49(+) T cells were reduced severely in the spleen, bone marrow, and liver, but not thymus, in mice doubly deficient for IFN regulatory factor-2 (IRF-2) and CD1d, in which the overall memory phenotype T cell population was contrastingly enlarged. Because a large fraction of Ly49(+) T cells coexpressed NK1.1 or NKG2D, the reduction of Ly49(+) T cells resulted indirectly in underrepresentation of NK1.1(+) or NKG2D(+) cells. Ly49(+) T cell deficiency was observed in IRF-2(-/-) mice additionally lacking IFN-α/ßR α-chain (IFNAR1) as severely as in IRF-2(-/-) mice, arguing against the involvement of the accelerated IFN-α/ß signals due to IRF-2 deficiency. Rather, mice lacking IFN-α/ßR alone also exhibited relatively milder Ly49(+) T cell reduction, and IL-2 could expand Ly49(+) T cells from IFNAR1(-/-), but not from IRF-2(-/-), spleen cells in vitro. These results together indicated that IRF-2 acted in Ly49(+) T cell development in a manner distinct from that of IFN-α/ß signals. The influence of IRF-2 deficiency on Ly49(+) memory phenotype T cells observed in this study suggested a unique transcriptional program for this T cell population among other NKR(+) T and memory phenotype T cells.
Assuntos
Antígenos CD1d/fisiologia , Diferenciação Celular/imunologia , Fator Regulador 2 de Interferon/fisiologia , Receptores de Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Antígenos CD1d/genética , Diferenciação Celular/genética , Memória Imunológica/genética , Memória Imunológica/imunologia , Imunofenotipagem/métodos , Fator Regulador 2 de Interferon/deficiência , Fator Regulador 2 de Interferon/genética , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Camundongos , Camundongos Knockout , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/patologiaRESUMO
A major task for the immune system is to secure powerful immune reactions while preserving self-tolerance. This process is particularly challenging for NK cells, for which tolerizing inhibitory receptors for self-MHC class I is both cross-reactive and expressed in an overlapping fashion between NK cells. We show in this study that during an education process, self-MHC class I molecules enrich for potentially useful and contract potentially dangerous NK cell subsets. These processes were quantitatively controlled by the expression level of the educating MHC class I allele, correlated with susceptibility to IL-15 and sensitivity to apoptosis in relevant NK cell subsets, and were linked to their functional education. Controlling the size of NK cell subsets with unique compositions of inhibitory receptors may represent one mechanism by which self-MHC class I molecules generate a population of tolerant NK cells optimally suited for efficient missing self-recognition.
Assuntos
Antígenos H-2/biossíntese , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Proliferação de Células , Células Cultivadas , Relação Dose-Resposta Imunológica , Feminino , Antígenos H-2/genética , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/deficiênciaRESUMO
In mice lacking IL-15, NK cell development is arrested at immature stages, providing an opportunity to investigate the earliest developing NK cells that would respond to IL-15. We show in this study that immature NK cells were present in the spleen as well as bone marrow (BM) and contained IL-15-high-responder cells. Thus, mature NK cells were generated more efficiently from IL-15(-/-) than from control donor cells in radiation BM chimeras, and the rate of IL-15-induced cell division in vitro was higher in NK cells in the spleen and BM from IL-15(-/-) mice than in those from wild-type mice. Phenotypically, NK cells developed in IL-15(-/-) mice up to the minor but discrete CD11b(-)CD27(+)DX5(hi)CD51(dull)CD127(dull)CD122(hi) stage, which contained the majority of Ly49G2(+) and D(+) NK cells both in the spleen and BM. Even among wild-type splenic NK cells, IL-15-induced proliferation was most prominent in CD11b(-)DX5(hi) cells. Notably, IL-15-mediated preferential expansion (but not conversion from Ly49(-) cells) of Ly49(+) NK cells was observed in vitro only for NK cells in the spleen. These observations indicated the uneven distribution of NK cells of different developing stages with variable IL-15 responsiveness in these lymphoid organs. Immature NK cells in the spleen may contribute, as auxiliaries to those in BM, to the mature NK cell compartment through IL-15-driven extramarrow expansion under steady-state or inflammatory conditions.
Assuntos
Diferenciação Celular/imunologia , Interleucina-15/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-15/deficiência , Interleucina-15/genética , Células Matadoras Naturais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/deficiência , Baço/citologia , Baço/crescimento & desenvolvimento , Baço/imunologiaRESUMO
NK cell responses are determined by signals received through activating and inhibitory cell surface receptors. Ly49H is an NK cell-specific activating receptor that accounts for the genetic resistance to murine CMV (MCMV). The Ly49H receptor has been shown to interact with two adaptor proteins (DAP12 and DAP10). In the context of MCMV infection, interaction of m157 (the MCMV-encoded ligand for Ly49H) with Ly49H results in activation of Ly49H-expressing NK cells. Chronic exposure of Ly49H with m157, however, induces tolerance in these same cells. The mechanism of this tolerance remains poorly understood. Using a transgenic mouse model, we demonstrate that induction of tolerance in Ly49H(+) NK cells by chronic exposure to m157, in vivo, requires signaling through the Ly49H adaptor protein DAP12, but not the DAP10 adaptor protein. Furthermore, mature Ly49H-expressing NK cells from wild-type mice can acquire a tolerant phenotype by 24 h posttransfer into a transgenic C57BL/6 mouse that expresses m157. The tolerant phenotype can be reversed, in vivo, if tolerant NK cells are transferred to mice that do not express the m157 protein. Thus, continuous activating receptor engagement can induce a transient tolerance in mature NK cells in vivo. These observations provide new insight into how activating receptor engagement shapes NK cell function and has important implications in how NK cells respond to tumors and during chronic viral infection.
Assuntos
Diferenciação Celular/imunologia , Tolerância Imunológica , Muromegalovirus/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/fisiologia , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Receptores Imunológicos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transferência Adotiva , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Introdução de Genes , Tolerância Imunológica/genética , Ligantes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
Ly49E is an unusual member of the Ly49 family that is expressed on fetal NK cells, epithelial T cells, and NKT cells, but not on resting adult NK cells. Ly49E(bgeo/bgeo) mice in which the Ly49E gene was disrupted by inserting a ß-geo transgene were healthy, fertile, and had normal numbers of NK and T cells in all organs examined. Their NK cells displayed normal expression of Ly49 and other NK cell receptors, killed tumor and MHC class I-deficient cells efficiently, and produced normal levels of IFN-γ. In heterozygous Ly49E(+/bgeo) mice, the proportion of epidermal T cells, NKT cells, and IL-2-activated NK cells that expressed Ly49E was about half that found in wild-type mice. Surprisingly, although splenic T cells rarely expressed Ly49E, IL-2-activated splenic T cells from Ly49E(bgeo/bgeo) mice were as resistant to growth in G418 as NK cells and expressed similar levels of ß-geo transcripts, suggesting that disruption of the Ly49E locus had increased its expression in these cells to the same level as that in NK cells. Importantly, however, the proportion of G418-resistant heterozygous Ly49E(+/bgeo) cells that expressed Ly49E from the wild-type allele was similar to that observed in control cells. Collectively, these findings demonstrate that Ly49E is not required for the development or homeostasis of NK and T cell populations or for the acquisition of functional competence in NK cells and provide compelling evidence that Ly49E is expressed in a probabilistic manner in adult NK cells and T cells.
Assuntos
Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/deficiência , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Senescência Celular/genética , Senescência Celular/imunologia , Técnicas de Introdução de Genes , Células Matadoras Naturais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossínteseRESUMO
Mouse CMV (MCMV) infection rapidly induces the proliferation of NK cells, which correlates with immunological protection. Whether NK cells primed during acute response against MCMV are maintained for the long term is not known. In this study, we used TcrdH2BeGFP mice in which maturing NK cells are genetically labeled with a pulse of very stable histone-2B-eGFP. In this system, we found that the reporter protein was diluted out upon NK cell division during acute MCMV infection. At the same time, mature NK cells in uninfected mice showed only very limited turnover in vivo. Three months after primary infection when MCMV latency was established, the majority of peripheral NK cells still displayed a higher record of proliferation than NK cells in mock-infected controls. This observation included both Ly49H(+) and Ly49H(-) NK cells. Conversely, naive NK cells did not show more proliferation after transfer into latently MCMV-infected mice than that after transfer into mock-infected control mice. This indicated that the observed alterations of the NK cell compartment in MCMV latency were "legacy" (i.e., resulting from prior events during the initial immune response). Together, these results suggest that antiviral immune responses induce sustained alterations of innate lymphocyte populations that extend far beyond the first days of acute infection.
Assuntos
Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Imunidade Inata/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Latência Viral/imunologia , Doença Aguda , Animais , Infecções por Citomegalovirus/patologia , Proteínas de Fluorescência Verde/genética , Histonas/genética , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muromegalovirus/genética , Muromegalovirus/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/deficiência , Subfamília A de Receptores Semelhantes a Lectina de Células NK/fisiologia , Latência Viral/genéticaRESUMO
In MHC class I-deficient hosts, natural killer (NK) cells are hyporesponsive to cross-linking of activation receptors. Functional competence requires engagement of a self-major histocompatability complex (MHC) class I-specific inhibitory receptor, a process referred to as "licensing." We previously suggested that licensing is developmentally determined in the bone marrow. In this study, we find that unlicensed mature MHC class I-deficient splenic NK cells show gain-of-function and acquire a licensed phenotype after adoptive transfer into wild-type (WT) hosts. Transferred NK cells produce WT levels of interferon-γ after engagement of multiple activation receptors, and degranulate at levels equivalent to WT NK cells upon coincubation with target cells. Only NK cells expressing an inhibitory Ly49 receptor specific for a cognate host MHC class I molecule show this gain-of-function. Therefore, these findings, which may be relevant to clinical bone marrow transplantation, suggest that neither exposure to MHC class I ligands during NK development in the BM nor endogenous MHC class I expression by NK cells themselves is absolutely required for licensing.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Transferência Adotiva , Animais , Transplante de Medula Óssea , Diferenciação Celular , Interferon gama/biossíntese , Interferon gama/imunologia , Células Matadoras Naturais/transplante , Camundongos , Camundongos Endogâmicos C57BL , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Imunologia de TransplantesRESUMO
Some mature natural killer (NK) cells cannot be inhibited by major histocompatibility complex (MHC) I molecules, either because they lack corresponding inhibitory receptors or because the host lacks the corresponding MHC I ligands for the receptors. Such NK cells nevertheless remain self-tolerant and exhibit a generalized hyporesponsiveness to stimulation through activating receptors. To address whether NK cell responsiveness is set only during the NK cell differentiation process, we transferred mature NK cells from wild-type (WT) to MHC I-deficient hosts or vice versa. Remarkably, mature responsive NK cells from WT mice became hyporesponsive after transfer to MHC I-deficient mice, whereas mature hyporesponsive NK cells from MHC I-deficient mice became responsive after transfer to WT mice. Altered responsiveness was evident among mature NK cells that had not divided in the recipient animals, indicating that the cells were mature before transfer and that alterations in activity did not require cell division. Furthermore, the percentages of NK cells expressing KLRG1, CD11b, CD27, and Ly49 receptors specific for H-2(b) were not markedly altered after transfer. Thus, the functional activity of mature NK cells can be reset when the cells are exposed to a changed MHC environment. These findings have important implications for how NK cell functions may be curtailed or enhanced in the context of disease.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Animais , Antígeno CD11b/biossíntese , Antígeno CD11b/imunologia , Diferenciação Celular , Divisão Celular , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Células Matadoras Naturais/transplante , Lectinas Tipo C , Camundongos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores Imunológicos/biossíntese , Receptores Imunológicos/imunologia , Tolerância a Antígenos Próprios/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Osteoclasts, multinucleated cells that resorb bone, play a key role in bone remodeling. Although immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling is critical for osteoclast differentiation, the significance of immunoreceptor tyrosine-based inhibitory motif (ITIM) has not been well understood. Here we report the function of Ly49Q, an Ly49 family member possessing an ITIM motif, in osteoclastogenesis. Ly49Q is selectively induced by receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) stimulation in bone marrow-derived monocyte/macrophage precursor cells (BMMs) among the Ly49 family of NK receptors. The knockdown of Ly49Q resulted in a significant reduction in the RANKL-induced formation of tartrate-resistance acid phosphatase (TRAP)-positive multinucleated cells, accompanied by a decreased expression of osteoclast-specific genes such as Nfatc1, Tm7sf4, Oscar, Ctsk, and Acp5. Osteoclastogenesis was also significantly impaired in Ly49Q-deficient cells in vitro. The inhibitory effect of Ly49Q-deficiency may be explained by the finding that Ly49Q competed for the association of Src-homology domain-2 phosphatase-1 (SHP-1) with paired immunoglobulin-like receptor-B (PIR-B), an ITIM-bearing receptor which negatively regulates osteoclast differentiation. Unexpectedly, Ly49Q deficiency did not lead to impaired osteoclast formation in vivo, suggesting the existence of a compensatory mechanism. This study provides an example in which an ITIM-bearing receptor functions as a positive regulator of osteoclast differentiation.
Assuntos
Diferenciação Celular , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Osteoclastos/citologia , Motivos de Aminoácidos , Animais , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Mutantes , NF-kappa B/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Ligante RANK/metabolismo , Interferência de RNARESUMO
Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for "missing self" recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an "educating impact" on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.
Assuntos
Regulação da Expressão Gênica , Genes MHC Classe I , Células Matadoras Naturais/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Animais , Separação Celular , Biologia Computacional , Simulação por Computador , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Modelos Estatísticos , SoftwareRESUMO
NKRP1 receptors were discovered more than 20 years ago, but due to a lack of appropriate reagents, our understanding of them has remained limited. Using a novel panel of mAbs that specifically recognize mouse NKRP1A, D, and F molecules, we report here that NKRP1D expression is limited to a subpopulation of NK cells, but in contrast to Ly49 receptors appears to be expressed in a normal codominant manner. NKRP1D(-) and NKRP1D(+) NK cells are functionally distinct, NKRP1D(+) cells showing reduced expression of various Ly49 receptors, elevated expression of CD94/NKG2 receptors, and higher IFN-gamma secretion and cytotoxicity than NKRP1D(-) cells. Furthermore, NKRP1D(+) NK cells were unable to kill transfected cells expressing high levels of Clr-b molecules, but readily killed MHC class-I-deficient blast cells that express only low levels of Clr-b. NKRP1A and NKRP1F were expressed at low levels on all splenic and bone marrow NK cells, but mAb-induced cross-linking of NKRP1A and NKRP1F caused no significant enhancement or inhibition of NK cell cytotoxicity and no detectable production of IFN-gamma. NKRP1A, D, and F expression could not be detected on NKT cells, all of which express NKRP1C, and although some activated T cells expressed NKRP1C and perhaps low levels of NKRP1A, no significant expression of NKRP1D or F could be detected. NKRP1 molecules expressed on NK cells or transfectants were down-regulated by cross-linking with mAbs or cell surface ligands, and using this phenomenon as a functional assay for NKRP1-ligand interaction revealed that NKRP1F can recognize CLR-x.
Assuntos
Regulação da Expressão Gênica/imunologia , Família Multigênica/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Animais , Anticorpos Monoclonais/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Sítios de Ligação de Anticorpos/imunologia , Ligação Competitiva/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica/genética , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica/genética , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/fisiologia , Ratos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Although it is well established that human NK cells are able to detect the absence of autologous HLA class I in vitro by virtue of inhibitory killer Ig-like receptors (KIR), direct evidence that KIR can mediate "missing self" recognition in vivo is lacking. To test this, we generated mice transgenic for a human KIR B-haplotype and HLA-Cw3 on a C57BL/6 background. NK cells in these mice expressed multiple KIR in a stochastic manner, including the HLA-Cw3-specific inhibitory receptor KIR2DL2. KIR and HLA transgenic mice rejected wild-type C57BL/6 spleen cells upon i.v. injection. This rejection was dependent on the presence of the KIR transgene in the host and on the absence of HLA-Cw3 from the injected target cells. Hence, the KIR transgene mediated "missing self" recognition in vivo. We anticipate that this KIR and HLA transgenic mouse will help shed light on KIR and HLA effects in disease and transplantation.
Assuntos
Apresentação de Antígeno/imunologia , Autoantígenos/metabolismo , Receptores KIR2DL2/fisiologia , Animais , Apresentação de Antígeno/genética , Autoantígenos/genética , Autoantígenos/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Antígenos HLA-C/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Receptores KIR2DL2/deficiência , Receptores KIR2DL2/genética , Baço/citologia , Baço/imunologia , Baço/metabolismo , Baço/transplante , Processos EstocásticosRESUMO
Uterine NK (uNK) cells are a prominent feature of the uterine mucosa and regulate placentation. NK cell activity is regulated by a balance of activating and inhibitory receptors, however the receptor repertoire of mouse uNK cells is unknown. We describe herein two distinct subsets of CD3(-)CD122(+) NK cells in the mouse uterus (comprising decidua and mesometrial lymphoid aggregate of pregnancy) at mid-gestation: a small subset indistinguishable from peripheral NK cells, and a larger subset that expresses NKp46 and Ly49 receptors, but not NK1.1 or DX5. This larger subset reacts with Dolichus biflores agglutinin, a marker of uNK cells in the mouse, and is adjacent to the invading trophoblast. By multiparametric analysis we show that the phenotype of uNK cells is unique and unprecedented in terms of adhesion, activation, and MHC binding potential. Thus, the Ly49 repertoire and the expression of other differentiation markers strikingly distinguish uNK cells from peripheral NK cells, suggesting that a selection process shapes the receptor repertoire of mouse uNK cells.