RESUMO
MicroRNAs (miRNAs) play important roles in initiation, development, progression and metastasis of tumors. MiR-342 has been reported as a tumor suppressor or an onco-miRNA based on functions or expression changes in various types of cancers. However, the biological roles and underlying molecular mechanisms of miR-342 in tumorigenesis remain largely unknown. Here, we found that miR-342 was expressed significantly less in a murine MS-K tumor cell line that showed riched blood vessels. Over-expression of miR-342 in MS-K cells inhibited cell proliferation, colony formation, reduced frequency of S phase population in vitro and suppressed tumor growth in vivo. Moreover, increasing miR-342 impeded blood vessels formation and accumulation of macrophages (CD11b+ ) in tumors. By bioinformatic analysis and dual-luciferase reporter assays, chemokine CXCL12 was identified as a direct target of miR-342. Restored Cxcl12 expression in MS-K-miR-342 cells could rescue cell proliferation in vitro. In MS-K-miR-342 tumor-infiltrated macrophages, expression of proangiogenic genes (Vegf-A and Thbs1) and M2-subtype macrophage markers (Cd163, Dectin1 and Ym1) was significantly down-regulated compared with controls. Moreover, lower level of Cxcl12 and its receptor Cxcr4 was observed in the macrophages of MS-K-miR-342 tumors, and MS-K-miR-342 derived miR-342, but not endogenous miR-342, might contribute to Cxcl12 suppression in TAM. These results suggest that miR-342 is involved in MS-K tumor growth as a tumor suppressor by targeting chemokine CXCL12.
Assuntos
Quimiocina CXCL12/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , MicroRNAs/metabolismo , Sarcoma/genética , Sarcoma/patologia , Animais , Apoptose , Sequência de Bases , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Camundongos , MicroRNAs/genéticaRESUMO
We previously demonstrated that IL-18 and CCL11 were highly expressed in an NFSA tumor cell line that showed limited angiogenesis and severe necrosis. However, IL-18 was not responsible for the immune cell accumulation and necrosis. Here, we attempted to clarify the relevance of CCL11 in angiogenesis and tumor formation. We established CCL11-overexpressing MS-K cell clones (MS-K-CCL11) to assess the role of CCL11 in immune cell accumulation and angiogenesis. The MS-K-CCL11 cells did not form tumors in mice. MS-K-CCL11-conditioned medium (CM) and recombinant CCL11 induced macrophage and eosinophil differentiation from bone marrow cells. The MS-K-CCL11-CM effectively recruited the differentiated eosinophils. Furthermore, the eosinophils damaged the MS-K, NFSA and endothelial cells in a dose-dependent manner. Administration of an antagonist of CCR3, a CCL11 receptor, to NFSA tumor-bearing mice restored the blood vessel formation and blocked the eosinophil infiltration into the NFSA tumors. Furthermore, other CCL11-overexpressing LM8 clones were established, and their tumor formation ability was reduced compared to the parental LM8 cells, accompanied by increased eosinophil infiltration, blockade of angiogenesis and necrosis. These results indicate that CCL11 was responsible for the limited angiogenesis and necrosis by inducing and attracting eosinophils in the tumors.
Assuntos
Quimiocina CCL11/imunologia , Fibrossarcoma/patologia , Granulócitos/imunologia , Neovascularização Patológica , Animais , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Eosinófilos/metabolismo , Fibrossarcoma/irrigação sanguínea , Camundongos , Naftalenos , Fenilalanina/análogos & derivados , Receptores CCR3/antagonistas & inibidoresRESUMO
We previously showed that interleukin (IL)-18 produced by NFSA cells induced the M1 type of macrophages in NFSA tumors, caused the destruction of endothelial cells in vitro and may have resulted in the necrosis of NFSA tumors by enhancing macrophage phagocytosis and cytotoxicity. However, the effect of IL-18 on blood vessel formation in vivo has not been elucidated. MS-K cells do not express il-18, and they form tumors with well-developed blood vessels. Here, we established IL-18-over-expressing MS-K cell clones (MS-K-IL-18) to address the roles of IL-18 in angiogenesis. The over-expression of IL-18 inhibited the proliferation rate of the MS-K-IL-18 cells in vitro and blood vessel formation in the MS-K-IL-18 tumors. Interestingly, CD14-positive cells from the MS-K-IL-18 tumor had up-regulated expression of the M1-type macrophage marker il-6 and down-regulated expression of interferon (ifn)-γ. Furthermore, FACS analysis showed more accumulation of CD11b+/CD80+ M1 macrophages in the MS-K-IL-18 tumors than in the parental MS-K tumor. Moreover, an in vitro coculture assay showed that MS-K-IL-18-conditioned medium (CM) stimulated macrophages to induce the apoptosis of endothelial cells. Cumulatively, our data showed that IL-18 inhibited tumor blood vessel formation in vivo.
Assuntos
Interleucina-18/farmacologia , Macrófagos/metabolismo , Neovascularização Patológica/metabolismo , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-18/uso terapêutico , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Neovascularização Patológica/tratamento farmacológicoRESUMO
Although a major function of B cells is to mediate humoral immunity by producing antigen-specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). However, the mechanism by which IL-10 is induced in B cells has not been fully elucidated. Here, we report that IκBNS , an inducible nuclear IκB protein, is important for Toll-like receptor (TLR)-mediated IL-10 production in B cells. Studies using IκB(NS) knockout mice revealed that the number of IL-10-producing B cells is reduced in IκB(NS)(-/-) spleens and that the TLR-mediated induction of cytoplasmic IL-10-positive cells and IL-10 secretion in B cells are impaired in the absence of IκB(NS). The impairment of IL-10 production by a lack of IκB(NS) was not observed in TLR-triggered macrophages or T-cell-receptor-stimulated CD4(+) CD25(+) T cells. In addition, IκB(NS)-deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL-10(+) CD138(+) plasmablasts. These results suggest that IκB(NS) is selectively required for IL-10 production in B cells responding to TLR signals, so defining an additional role for IκB(NS) in the control of the B-cell-mediated immune responses.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas I-kappa B/metabolismo , Interleucina-10/biossíntese , Receptores Toll-Like/metabolismo , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Proteínas I-kappa B/deficiência , Proteínas I-kappa B/genética , Interleucina-10/genética , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Baço , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The autoantibodies (auto-Abs) that are a hallmark of neonatally thymectomized (NTx) mice with autoimmune gastritis (AIG) have been poorly explored. We investigated their immune significance using B cell-deficient (B(-)) mice and found that B(-) mice are totally resistant to AIG but become susceptible to AIG after receiving bone marrow cells from B(+) mice. This susceptibility is most likely caused by the production of auto-Abs by B cells because B(-) pups also became susceptible to AIG when nourished by an AIG dam producing auto-Abs of the IgG class during the suckling period. NTx B(-) mice receiving purified IgG auto-Abs at this developmental stage similarly developed AIG. Auto-Abs probably act on antigen handling for antigen presentation because the treatment of NTx B(+) mice with anti-FcγR Abs prevented the development of AIG. Auto-Abs are indispensable for AIG development but are not sufficient because auto-Ab treatment did not increase AIG incidence in NTx B(+) mice above the baseline.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Gastrite/imunologia , Receptores de IgG/imunologia , Transferência Adotiva , Animais , Animais Recém-Nascidos , Linfócitos B , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Receptores de IgG/antagonistas & inibidores , TimectomiaRESUMO
If Bcl11b activity is compromised, CD4(+)CD8(+) double-positive (DP) thymocytes produce a greatly increased fraction of innate CD8(+) single-positive (SP) cells highly producing IFN-γ, which are also increased in mice deficient of genes such as Itk, Id3 and NF-κB1 that affect TCR signaling. Of interest, the increase in the former two is due to the bystander effect of IL-4 that is secreted by promyelocytic leukemia zinc finger-expressing NKT and γδT cells whereas the increase in the latter is cell intrinsic. Bcl11b zinc-finger proteins play key roles in T cell development and T cell-mediated immune response likely through TCR signaling. We examined thymocytes at and after the DP stage in Bcl11b (F/S826G) CD4cre, Bcl11b (F/+) CD4cre and Bcl11b (+/S826G) mice, carrying the allele that substituted serine for glycine at the position of 826. Here we show that Bcl11b impairment leads to an increase in the population of TCRαß(high)CD44(high)CD122(high) innate CD8SP thymocytes, together with two different developmental abnormalities: impaired positive and negative selection accompanying a reduction in the number of CD8SP cells, and developmental arrest of NKT cells at multiple steps. The innate CD8SP thymocytes express Eomes and secrete IFN-γ after stimulation with PMA and ionomycin, and in this case their increase is not due to a bystander effect of IL-4 but cell intrinsic. Those results indicate that Bcl11b regulates development of different thymocyte subsets at multiple stages and prevents an excess of innate CD8SP thymocytes.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Receptores de Hialuronatos/metabolismo , Proteínas Inibidoras de Diferenciação/genética , Interferon gama/biossíntese , Subunidade beta de Receptor de Interleucina-2/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/genética , Células T Matadoras Naturais/imunologia , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais/imunologiaRESUMO
We investigated the expansion rate of CD4(+) memory T cells using a newly developed in vivo system. Neonatal thymectomy abrogates the subsequent production of T cells and induces autoimmune gastritis (AIG) by the activation of CD4(+) T cells; this disease was transferred into athymic nude mice through the inoculation of splenic CD4(+) memory T cells. The transferred CD4(+) T cells increased logarithmically in number during the first 2months in the spleen of the recipients. The serial transfer of these splenocytes at two-month intervals revealed that the numbers of the AIG-transferable generations were inversely correlated with the age of the first AIG donors. The duration of the AIG-promoting capacity of CD4(+) T cells under continuous antigenic stimulation in vivo was approximately equivalent-one and a half years. These results indicate that there exists an intrinsic population doubling limit in memory CD4(+) T cells similar to that of self-renewing naïve ones.
Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica , Envelhecimento , Animais , Linfócitos T CD4-Positivos/citologia , Feminino , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptores de Antígenos de Linfócitos T/imunologia , Baço/imunologia , TimectomiaRESUMO
Neonatal thymectomy (NTx) induces autoimmune gastritis (AIG) in BALB/c mice, a model for human type A chronic atrophic gastritis, but not in DBA/2 mice and rarely in CDF1 mice (a hybrid of BALB/c and DBA/2 mice). The aim of this study was to clarify the mechanisms of AIG-resistance in mice bearing the dominant trait of DBA/2. Linkage groups associated with, and cells related to AIG resistance were examined with CDF1-BALB/c backcrosses. Intracellular staining and flow-cytometric bead array for several cytokines were performed on NTx BALB/c mice and NTx DBA/2-chimeric BALB/c mice receiving DBA/2-bone marrow cells. In NTx BALB/c mice, IFN-γ-secreting CD4(+) T cells were increased, but not in NTx DBA/2 mice. Because Vß6(+) T cell-bearing mice of half of their backcrosses developed AIG, but the other half of Vß6(+) T cell-negative mice developed scarcely, resistance for AIG generation is associated with the presence of the Mls-1a locus on chromosome 1 in DBA/2 mice, which deletes Vß6(+) T cells. NTx DBA/2-chimera BALB/c mice showed dominant production of IL-10 and resistance for AIG, although the deletion of Vß6(+) T cells was found not to be a cause of AIG-resistance from Mls-1a locus segregation experiments. Although NTx DBA/2-chimeric BALB/c mice did not suffer from AIG, they brought immediate precursors of T cells for AIG. It is concluded that DBA/2 mice generate bone marrow-derived cells that produce anti-inflammatory cytokines to prevent the activation of AIG-T cells.
Assuntos
Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Células da Medula Óssea , Cromossomos de Mamíferos/genética , Gastrite/genética , Gastrite/imunologia , Genes Dominantes/genética , Linfócitos T/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Quimera/genética , Citocinas/metabolismo , Citocinas/fisiologia , Modelos Animais de Doenças , Resistência à Doença/genética , Feminino , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA/genética , Linfócitos T/metabolismoRESUMO
Murine MS-K and NFSA cell lines formed tumor after inoculation into mouse and both cell lines expressed high level of vascular endothelial growth factor-A (vegf-A) and produced same level of VEGF-A. However, poor blood vessel formation, and necrosis was significantly observed in NFSA-tumor, contrary to well-developed blood vessel formation in MS-K tumor. The microarray analysis showed high expression of fibroblast growth factor-10 (fgf-10) in MS-K than NFSA. In this report, the role of fgf-10 on tumor growth was studied. MS-K enhanced more proliferation of endothelial cells by direct co-culture than NFSA, and rFGF10 supported the proliferation of HUVEC in combination with VEGF-A. fgf-10-knocked down MS-K, MS-K (fgf-10-KD), proliferated slower in vitro and the tumorigenicity of them was also slower than control. The blood vessel formation in these MS-K (fgf-10-KD) clones was reduced compared with the MS-K (normal). qPCR analysis showed the suppression of vegf-A, vegf-C and fgfr-1-expression in the MS-K (fgf-10-KD) clones. Taken together, these results indicated that FGF10, which was produced from tumor cells, was essential for the proliferation of tumor cell itself and also supports proliferation of endothelial cells. Thus, FGF10 plays an important role for tumor growth by both paracrine and autocrine manner.
Assuntos
Carcinogênese/metabolismo , Fator 10 de Crescimento de Fibroblastos/metabolismo , Neovascularização Patológica/metabolismo , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Fator 10 de Crescimento de Fibroblastos/genética , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Camundongos , Dados de Sequência Molecular , Transplante de Neoplasias , Neovascularização Patológica/patologiaRESUMO
Inoculation of mice with the murine NFSA cell line caused the formation of large tumors with necrotic tumor cores. FACS analysis revealed accumulations of CD11b(+) cells in the tumors. Microarray analysis indicated that the NFSA cells expressed a high level of the pro-inflammatory factor interleukin-18 (il-18), which is known to play a critical role in macrophages. However, little is known about the physiological function of IL-18-stimulated macrophages. Here, we provide direct evidence that IL-18 enhances the phagocytosis of RAW264 cells and peritoneal macrophages, accompanied by the increased expression of tumor necrosis factor (tnf-α), interleukin-6 (il-6) and inducible nitric oxide synthase (Nos2). IL-18-stimulated RAW264 cells showed an enhanced cytotoxicity to endothelial F-2 cells via direct cell-to-cell interaction and the secretion of soluble mediators. Taken together, our results demonstrate that tumor-derived IL-18 plays an important role in the phagocytosis of macrophages and that IL-18-stimulated macrophages may damage tumor endothelial cells.
Assuntos
Interleucina-18/imunologia , Macrófagos/imunologia , Neoplasias Experimentais/imunologia , Animais , Antígeno CD11b/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Células Endoteliais/imunologia , Expressão Gênica , Interleucina-6/genética , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C3H , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/genética , Óxido Nítrico Sintase Tipo II/genética , Fagocitose , Proteínas Recombinantes/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Urodele amphibians are thought to have poorer immune responses than evolutionary more ancestral vertebrate classes, such as bony fish. We investigated skin graft rejection and transplantation immunity in Urodele amphibians, Japanese newts, and Asiatic salamanders, and compared these findings to those from transplants in several species of frogs. The skin grafts used in this study were either allogeneic or xenogeneic. The mean survival time of the first set of allografts at 20°C was approximately 60 days for chronic responses in Urodela and 20 days for acute responses in Anura. As the graft survival times of urodeles were significantly longer than those of anurans, even when urodeles were repeatedly grafted from identical donors, there appear to be substantial differences in transplantation immunity between Urodela and Anura. These slow responses in Urodela may not be accompanied by the expansion of cytotoxic T cells, as observed in fish and anuran species, which are known to have functional major histocompatibility complex (MHC)-class I systems. In our study, approximately five histo-incompatible immunogenic components were found to be involved in chronic responses in newts. Similar chronic responses were also observed in xenograft rejection in newts. In contrast, xenografts were rejected in frogs due to an accelerated acute response, possibly involving natural killer cells. Our findings that some anti-allogeneic components appear to be shared with xenogeneic components indicate that the diversification of the acquired immune system is poorly developed in Urodela.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Pele/imunologia , Transplante Heterólogo/imunologia , Urodelos/imunologia , Animais , Cicatrização/imunologiaRESUMO
The thymus involutes after puberty, although the mechanism by which this process occurs remains poorly understood. The profile of thymic involution, which is inversely correlated with an increase in peripheral T cells, may indicate that the accumulation of T cells in the periphery is related to thymic atrophy. In this study, it was shown that the prevention of T cell generation delayed the initiation of thymic involution. The activation of T cells increased the serum concentration of glucocorticoid (GC) and thymic involution, which was completely prevented by adrenalectomy. In the adrenals of growing mice, the activity of the zona fasciculata, which produces GC, increased and plateaued by the weaning period; however, the zona reticularis (ZR), which produces dehydroepiandrosterone (DHEA) that has anti-GC actions, started to decline just before puberty. Thymic atrophy was preceded by the infiltration of activated T cells into the ZR and by the loss of ZR cells. Thus, T cells are involved in thymic involution, a process which was retarded by DHEA administration, through an increase in GC activity due to ZR cell-killing.
Assuntos
Puberdade/fisiologia , Linfócitos T/fisiologia , Timo/patologia , Zona Reticular/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Atrofia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/fisiologia , Desidroepiandrosterona/fisiologia , Glucocorticoides/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Timo/fisiologia , Zona Reticular/patologiaRESUMO
IκBNS has been identified as a member of the IκB family of NF-κB inhibitors, which undergoes induction upon TCR signaling. Mice carrying a targeted gene disruption of IκBNS demonstrate dysregulation of cytokines in T cells, macrophages, and dendritic cells. IκBNS mediates both positive and negative gene regulation, depending on individual cell type and/or cytokine. In this study, we demonstrate an additional role for IκBNS in the B cell lineage. B cells from IκBNS knockout (KO) mice were impaired in proliferative responses to LPS and anti-CD40. IgM and IgG3 Igs were drastically reduced in the serum of IκBNS KO mice, although IκBNS KO B cells exhibited a higher level of surface IgM than that found in wild-type mice. Switching to IgG3 was significantly reduced in IκBNS KO B cells. The in vitro induction of plasma cell development demonstrated that progression to Ab-secreting cells was impaired in IκBNS KO B cells. In agreement with this finding, the number of Ab-secreting cells in the spleens of IκBNS KO mice was reduced and production of Ag-specific Igs was lower in IκBNS KO mice after influenza infection as compared with wild-type mice. Additionally, IκBNS KO mice lacked B1 B cells and exhibited a reduction in marginal zone B cells. Thus, IκBNS significantly impacts the development and functions of B cells and plasma cells.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Proteínas/imunologia , Animais , Linfócitos B/citologia , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/citologia , Plasmócitos/imunologia , Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The murine sarcoma cell line MS-K was previously established as a Ki-ras-positive cell line. Inoculation of this cell line under the flank of C3H/HeN mice results in the growth of large tumors with well-developed blood vessels within day 30 of transplantation without any metastasis because MS-K cells produce vascular endothelial growth factor (VEGF). To elucidate the role of VEGF in tumor formation in vivo, stable vegf-knockdown-MS-K clones were obtained using plasmid-based knockdown vectors. Interestingly, tumorigenesis was completely suppressed in a vegf-A-knockdown-MS-K clone [designated MS-K (A-KD)]. Proliferation and colony formation capacity of the MS-K (A-KD) cells in a semi-solid medium under low serum conditions was significantly lower than that of control MS-K (SCR) cells; however, the expression of vegf-receptor 1 (vegf-r-1) was not changed. Addition of the recombinant VEGF-A(165) partially restored the colony formation capacity of MS-K (A-KD) cells and caused the phosphorylation of VEGF-r-1 (Flt-1) in MS-K (Normal) cells. Furthermore, tumorigenicity of the vegf-r-1-knockdown-MS-K clone [designated MS-K (R1-KD)] had obviously delayed or strongly suppressed compared with the MS-K (Normal). These results indicate that Vascular endothelial growth factor-A, produced from MS-K, acts as a growth factor for MS-K cells itself and supports tumor formation in vivo by inducing the blood vessel formation.
Assuntos
Técnicas de Silenciamento de Genes , Sarcoma/genética , Sarcoma/metabolismo , Fatores de Crescimento do Endotélio Vascular/genética , Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C3H , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Sarcoma/patologia , Carga Tumoral/genética , Fator A de Crescimento do Endotélio Vascular/sangue , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(D,L-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp100(25-33) and TRP2(180-188), were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freund's adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2(180-188) and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.
Assuntos
Ácido Láctico/química , Nanopartículas/química , Peptídeos/imunologia , Ácido Poliglicólico/química , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos/química , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Células Dendríticas/imunologia , Citometria de Fluxo , Espectrometria de Massas , Camundongos , Peptídeos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Espectrometria de Massas em TandemRESUMO
The alphabeta TCR has recently been suggested to function as an anisotropic mechanosensor during immune surveillance, converting mechanical energy into a biochemical signal upon specific peptide/MHC ligation of the alphabeta clonotype. The heterodimeric CD3epsilongamma and CD3epsilondelta subunits, each composed of two Ig-like ectodomains, form unique side-to-side hydrophobic interfaces involving their paired G-strands, rigid connectors to their respective transmembrane segments. Those dimers are laterally disposed relative to the alphabeta heterodimer within the TCR complex. In this paper, using structure-guided mutational analysis, we investigate the functional consequences of a striking asymmetry in CD3gamma and CD3delta G-strand geometries impacting ectodomain shape. The uniquely kinked conformation of the CD3gamma G-strand is crucial for maximizing Ag-triggered TCR activation and surface TCR assembly/expression, offering a geometry to accommodate juxtaposition of CD3gamma and TCR beta ectodomains and foster quaternary change that cannot be replaced by the isologous CD3delta subunit's extracellular region. TCRbeta and CD3 subunit protein sequence analyses among Gnathostomata species show that the Cbeta FG loop and CD3gamma subunit coevolved, consistent with this notion. Furthermore, restoration of T cell activation and development in CD3gamma(-/-) mouse T lineage cells by interspecies replacement can be rationalized from structural insights on the topology of chimeric mouse/human CD3epsilondelta dimers. Most importantly, our findings imply that CD3gamma and CD3delta evolved from a common precursor gene to optimize peptide/MHC-triggered alphabeta TCR activation.
Assuntos
Complexo CD3/química , Complexo CD3/fisiologia , Multimerização Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Animais , Complexo CD3/genética , Evolução Molecular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Ovinos , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric alphabeta, CD3 epsilon gamma, CD3 epsilon delta, and CD3 zeta zeta subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3 epsilon lobe that they approach diagonally, adjacent to the lever-like C beta FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3 epsilon and CD3 gamma in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force ( approximately 50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.
Assuntos
Mecanotransdução Celular , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Complexo CD3/imunologia , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Conformação Molecular , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Triggering of the TCR by cognate peptide/MHC ligands induces expression of I kappa BNS, a member of the I kappa B family of NF-kappaB inhibitors whose expression is associated with apoptosis of immature thymocytes. To understand the role of I kappa BNS in TCR triggering, we created a targeted disruption of the I kappa BNS gene. Surprisingly, mice lacking I kappa BNS show normal thymic progression but both thymocytes and T cells manifest reduced TCR-stimulated proliferation. Moreover, I kappa BNS knockout thymocytes and T cells produce significantly less IL-2 and IFN-gamma than wild-type cells. Transfection analysis demonstrates that I kappa BNS and c-Rel individually increase IL-2 promoter activity. The effect of I kappa BNS on the IL-2 promoter, unlike c-Rel, is dependent on the NF-kappaB rather than the CD28RE site; mutation of the NF-kappaB site extinguishes the induction of transcription by I kappa BNS in transfectants and prevents association of I kappa BNS with IL-2 promoter DNA. Microarray analyses confirm the reduction in IL-2 production and some IFN-gamma-linked transcripts in I kappa BNS knockout T cells. Collectively, our findings demonstrate that I kappa BNS regulates production of IL-2 and other cytokines induced via "strong" TCR ligation.
Assuntos
Citocinas/biossíntese , Marcação de Genes , Proteínas/antagonistas & inibidores , Proteínas/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Citocinas/antagonistas & inibidores , Interleucina-2/biossíntese , Interleucina-2/genética , Interleucina-2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Isoantígenos/imunologia , Ligantes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/genética , Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismo , Transfecção , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
CD3epsilongamma and CD3epsilondelta are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal beta-strands that are linked to individual subunit membrane proximal stalk segments. CD3epsilon, CD3gamma, and CD3delta stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal beta-strand, we created a CD3gamma double mutant CD3gamma(C82S/C85S) and a CD3gamma beta-strand triple mutant CD3gamma(Q76S/Y78A/Y79A) for use in retroviral transduction of lymphoid progenitors for comparison with CD3gammawt. Although both mutant CD3gamma molecules reduced association with CD3epsilon in CD3epsilongamma heterodimers, CD3gamma(Q76S/Y78A/Y79A) abrogated surface TCR expression whereas CD3gamma(C82S/C85S) did not. Furthermore, CD3gamma(C82S/C85S) rescued thymic development in CD3gamma(-/-) fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3gamma(C82S/C85S) transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3gamma(-/-) thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3gammawt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3epsilongamma, as evidenced by the loss of reactivity with CD3gamma N terminus-specific antisera. Single histidine substitution of either CD3gamma stalk cysteine failed to restore CD3epsilongamma association and conformation in transient COS-7 cell transfection studies. Thus, CD3gamma(C82) and CD3gamma(C85) residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3epsilon(C80) and CD3epsilon(C83). The implications of these results for CD3epsilongamma and TCR structure and signaling function are discussed.
Assuntos
Complexo CD3/imunologia , Transdução de Sinais/imunologia , Timo/crescimento & desenvolvimento , Substituição de Aminoácidos/imunologia , Animais , Complexo CD3/genética , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto/imunologia , Técnicas de Cultura de Órgãos , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Receptores de Antígenos de Linfócitos T/imunologia , Retroviridae , Timo/imunologia , Transdução GenéticaRESUMO
The deletion of CD4- and CD8-double-positive (DP) cells in the thymus after treatment with anti-CD3 antibodies has long been considered as a useful model for clonal deletion during T cell development, although it was reported that DP cell death was not observed in neonates where self-tolerance should be developing. We dealt with the cellular basis of this enigmatic phenomenon in this report. Due to the similar susceptibility to the antibody-treatment in vitro between neonatal and adult thymocytes, critical factors may be outside rather than within the thymus. Indeed, newborn thymus lobes transplanted into recipients of different ages showed an increased susceptibility to the thymo-toxicity as the age of the recipient increased. The thymo-toxicity seems to be based on the adrenal function of glucocorticoid (GC) synthesis, because administration of an inhibitor of GC synthesis significantly reduced the DP cell death by the antibody-treatment. Finally, adrenalectomy completely prevented DP cell death by anti-CD3 antibodies in adult mice. Therefore, the thymocyte death by anti-CD3 antibodies in vivo may not be due to the T cellreceptor mediated selection in the thymus.