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1.
J Exp Med ; 215(1): 319-336, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29263218

RESUMO

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with constitutively activated mitogen-activated protein kinase (MAPK) pathway signaling. Approximately 60% of LCH patients harbor somatic BRAFV600E mutations localizing to CD207+ DCs within lesions. However, the mechanisms driving BRAFV600E+ LCH cell accumulation in lesions remain unknown. Here we show that sustained extracellular signal-related kinase activity induced by BRAFV600E inhibits C-C motif chemokine receptor 7 (CCR7)-mediated DC migration, trapping DCs in tissue lesions. Additionally, BRAFV600E increases expression of BCL2-like protein 1 (BCL2L1) in DCs, resulting in resistance to apoptosis. Pharmacological MAPK inhibition restores migration and apoptosis potential in a mouse LCH model, as well as in primary human LCH cells. We also demonstrate that MEK inhibitor-loaded nanoparticles have the capacity to concentrate drug delivery to phagocytic cells, significantly reducing off-target toxicity. Collectively, our results indicate that MAPK tightly suppresses DC migration and augments DC survival, rendering DCs in LCH lesions trapped and resistant to cell death.


Assuntos
Movimento Celular/fisiologia , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Histiocitose de Células de Langerhans/metabolismo , Células de Langerhans/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Animais , Apoptose/fisiologia , Histiocitose de Células de Langerhans/patologia , Humanos , Células de Langerhans/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação/fisiologia , Fagocitose/fisiologia
2.
Immunity ; 44(4): 924-38, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27096321

RESUMO

Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.


Assuntos
Antígenos CD/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Cadeias alfa de Integrinas/metabolismo , Melanoma Experimental/imunologia , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/farmacologia , Animais , Apresentação de Antígeno/imunologia , Linhagem Celular Tumoral , Células Dendríticas/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout
3.
Nat Immunol ; 16(10): 1060-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26343536

RESUMO

Treatment with ionizing radiation (IR) can lead to the accumulation of tumor-infiltrating regulatory T cells (Treg cells) and subsequent resistance of tumors to radiotherapy. Here we focused on the contribution of the epidermal mononuclear phagocytes Langerhans cells (LCs) to this phenomenon because of their ability to resist depletion by high-dose IR. We found that LCs resisted apoptosis and rapidly repaired DNA damage after exposure to IR. In particular, we found that the cyclin-dependent kinase inhibitor CDKN1A (p21) was overexpressed in LCs and that Cdkn1a(-/-) LCs underwent apoptosis and accumulated DNA damage following IR treatment. Wild-type LCs upregulated major histocompatibility complex class II molecules, migrated to the draining lymph nodes and induced an increase in Treg cell numbers upon exposure to IR, but Cdkn1a(-/-) LCs did not. Our findings suggest a means for manipulating the resistance of LCs to IR to enhance the response of cutaneous tumors to radiotherapy.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células de Langerhans/efeitos da radiação , Radiação Ionizante , Linfócitos T Reguladores/efeitos da radiação , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Citometria de Fluxo , Camundongos , Análise em Microsséries , Reação em Cadeia da Polimerase , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Regulação para Cima
4.
J Nucl Med ; 56(8): 1272-7, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26112022

RESUMO

UNLABELLED: Tumor-associated macrophages (TAMs) are increasingly investigated in cancer immunology and are considered a promising target for better and tailored treatment of malignant growth. Although TAMs also have high diagnostic and prognostic value, TAM imaging still remains largely unexplored. Here, we describe the development of reconstituted high-density lipoprotein (rHDL)-facilitated TAM PET imaging in a breast cancer model. METHODS: Radiolabeled rHDL nanoparticles incorporating the long-lived positron-emitting nuclide (89)Zr were developed using 2 different approaches. The nanoparticles were composed of phospholipids and apolipoprotein A-I (apoA-I) in a 2.5:1 weight ratio. (89)Zr was complexed with deferoxamine (also known as desferrioxamine B, desferoxamine B), conjugated either to a phospholipid or to apoA-I to generate (89)Zr-PL-HDL and (89)Zr-AI-HDL, respectively. In vivo evaluation was performed in an orthotopic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo histologic analysis of tumor tissues to assess regional distribution of (89)Zr radioactivity was also performed. Fluorescent analogs of the radiolabeled agents were used to determine cell-targeting specificity using flow cytometry. RESULTS: The phospholipid- and apoA-I-labeled rHDL were produced at 79% ± 13% (n = 6) and 94% ± 6% (n = 6) radiochemical yield, respectively, with excellent radiochemical purity (>99%). Intravenous administration of both probes resulted in high tumor radioactivity accumulation (16.5 ± 2.8 and 8.6 ± 1.3 percentage injected dose per gram for apoA-I- and phospholipid-labeled rHDL, respectively) at 24 h after injection. Histologic analysis showed good colocalization of radioactivity with TAM-rich areas in tumor sections. Flow cytometry revealed high specificity of rHDL for TAMs, which had the highest uptake per cell (6.8-fold higher than tumor cells for both DiO@Zr-PL-HDL and DiO@Zr-AI-HDL) and accounted for 40.7% and 39.5% of the total cellular DiO@Zr-PL-HDL and DiO@Zr-AI-HDL in tumors, respectively. CONCLUSION: We have developed (89)Zr-labeled TAM imaging agents based on the natural nanoparticle rHDL. In an orthotopic mouse model of breast cancer, we have demonstrated their specificity for macrophages, a result that was corroborated by flow cytometry. Quantitative macrophage PET imaging with our (89)Zr-rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment.


Assuntos
Lipoproteínas HDL/química , Nanopartículas/química , Tomografia por Emissão de Pósitrons , Radioisótopos/química , Zircônio/química , Animais , Modelos Animais de Doenças , Citometria de Fluxo , Macrófagos/metabolismo , Camundongos , Nanotecnologia , Prognóstico , Compostos Radiofarmacêuticos , Distribuição Tecidual , Tomografia Computadorizada por Raios X
5.
Genes Dev ; 27(17): 1868-85, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24013501

RESUMO

The p53 tumor suppressor is a transcription factor that mediates varied cellular responses. The C terminus of p53 is subjected to multiple and diverse post-translational modifications. An attractive hypothesis is that differing sets of combinatorial modifications therein determine distinct cellular outcomes. To address this in vivo, a Trp53(ΔCTD/ΔCTD) mouse was generated in which the endogenous p53 is targeted and replaced with a truncated mutant lacking the C-terminal 24 amino acids. These Trp53(ΔCTD/ΔCTD) mice die within 2 wk post-partum with hematopoietic failure and impaired cerebellar development. Intriguingly, the C terminus acts via three distinct mechanisms to control p53-dependent gene expression depending on the tissue. First, in the bone marrow and thymus, the C terminus dampens p53 activity. Increased senescence in the Trp53(ΔCTD/ΔCTD) bone marrow is accompanied by up-regulation of Cdkn1 (p21). In the thymus, the C-terminal domain negatively regulates p53-dependent gene expression by inhibiting promoter occupancy. Here, the hyperactive p53(ΔCTD) induces apoptosis via enhanced expression of the proapoptotic Bbc3 (Puma) and Pmaip1 (Noxa). In the liver, a second mechanism prevails, since p53(ΔCTD) has wild-type DNA binding but impaired gene expression. Thus, the C terminus of p53 is needed in liver cells at a step subsequent to DNA binding. Finally, in the spleen, the C terminus controls p53 protein levels, with the overexpressed p53(ΔCTD) showing hyperactivity for gene expression. Thus, the C terminus of p53 regulates gene expression via multiple mechanisms depending on the tissue and target, and this leads to specific phenotypic effects in vivo.


Assuntos
Regulação da Expressão Gênica , Genes p53/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Senescência Celular/genética , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Técnicas de Introdução de Genes , Crescimento e Desenvolvimento/genética , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Deleção de Sequência/genética , Timócitos/citologia , Timócitos/metabolismo , Fatores de Tempo
6.
Nature ; 467(7312): 214-7, 2010 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-20829794

RESUMO

Dendritic cells serve a key function in host defence, linking innate detection of microbes to activation of pathogen-specific adaptive immune responses. Whether there is cell-intrinsic recognition of human immunodeficiency virus (HIV) by host innate pattern-recognition receptors and subsequent coupling to antiviral T-cell responses is not yet known. Dendritic cells are largely resistant to infection with HIV-1, but facilitate infection of co-cultured T-helper cells through a process of trans-enhancement. Here we show that, when dendritic cell resistance to infection is circumvented, HIV-1 induces dendritic cell maturation, an antiviral type I interferon response and activation of T cells. This innate response is dependent on the interaction of newly synthesized HIV-1 capsid with cellular cyclophilin A (CYPA) and the subsequent activation of the transcription factor IRF3. Because the peptidylprolyl isomerase CYPA also interacts with HIV-1 capsid to promote infectivity, our results indicate that capsid conformation has evolved under opposing selective pressures for infectivity versus furtiveness. Thus, a cell-intrinsic sensor for HIV-1 exists in dendritic cells and mediates an antiviral immune response, but it is not typically engaged owing to the absence of dendritic cell infection. The virulence of HIV-1 may be related to evasion of this response, the manipulation of which may be necessary to generate an effective HIV-1 vaccine.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunidade Inata , Proteínas do Capsídeo/imunologia , Linhagem Celular , Ciclofilina A/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Ativação Linfocitária , Monócitos/citologia , Linfócitos T/imunologia
7.
Genes Dev ; 23(17): 2016-32, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19656801

RESUMO

PAZ/PIWI domain (PPD) proteins carrying small RNAs (sRNAs) function in gene and genome regulation. The ciliate Tetrahymena thermophila encodes numerous PPD proteins exclusively of the Piwi clade. We show that the three Tetrahymena Piwi family proteins (Twis) preferentially expressed in growing cells differ in their genetic essentiality and subcellular localization. Affinity purification of all eight distinct Twi proteins revealed unique properties of their bound sRNAs. Deep sequencing of Twi-bound and total sRNAs in strains disrupted for various silencing machinery uncovered an unanticipated diversity of 23- to 24-nt sRNA classes in growing cells, each with distinct genetic requirements for accumulation. Altogether, Twis distinguish sRNAs derived from loci of pseudogene families, three types of DNA repeats, structured RNAs, and EST-supported loci with convergent or paralogous transcripts. Most surprisingly, Twi7 binds complementary strands of unequal length, while Twi10 binds a specific permutation of the guanosine-rich telomeric repeat. These studies greatly expand the structural and functional repertoire of endogenous sRNAs and RNPs.


Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Protozoários/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Animais , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/química , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Ligação Proteica , RNA de Protozoário/química , Análise de Sequência de RNA , Proteína 1 Relacionada a Twist/metabolismo
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