RESUMO
We report a new immunodeficiency disorder in mice caused by a viable hypomorphic mutation of Snrnp40, an essential gene encoding a subunit of the U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome. Snrnp40 is ubiquitous but strongly expressed in lymphoid tissue. Homozygous mutant mice showed hypersusceptibility to infection by murine cytomegalovirus and multiple defects of lymphoid development, stability and function. Cell-intrinsic defects of hematopoietic stem cell differentiation also affected homozygous mutants. SNRNP40 deficiency in primary hematopoietic stem cells or T cells or the EL4 cell line increased the frequency of splicing errors, mostly intron retention, in several hundred messenger RNAs. Altered expression of proteins associated with immune cell function was also observed in Snrnp40-mutant cells. The immunological consequences of SNRNP40 deficiency presumably result from cumulative, moderate effects on processing of many different mRNA molecules and secondary reductions in the expression of critical immune proteins, yielding a syndromic immune disorder.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Infecções por Herpesviridae/imunologia , Síndromes de Imunodeficiência/imunologia , Muromegalovirus/fisiologia , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Spliceossomos/metabolismo , Linfócitos T/fisiologia , Alelos , Animais , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Suscetibilidade a Doenças , Infecções por Herpesviridae/genética , Síndromes de Imunodeficiência/genética , Linfopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U5/genéticaRESUMO
Signals emanating from the T-cell receptor (TCR), co-stimulatory receptors, and cytokine receptors each influence CD8 T-cell fate. Understanding how these signals respond to homeostatic and microenvironmental cues can reveal new ways to therapeutically direct T-cell function. Through forward genetic screening in mice, we discover that loss-of-function mutations in LDL receptor-related protein 10 (Lrp10) cause naive and central memory CD8 T cells to accumulate in peripheral lymphoid organs. Lrp10 encodes a conserved cell surface protein of unknown immunological function. T-cell activation induces Lrp10 expression, which post-translationally suppresses IL7 receptor (IL7R) levels. Accordingly, Lrp10 deletion enhances T-cell homeostatic expansion through IL7R signaling. Lrp10-deficient mice are also intrinsically resistant to syngeneic tumors. This phenotype depends on dense tumor infiltration of CD8 T cells, which display increased memory cell characteristics, reduced terminal exhaustion, and augmented responses to immune checkpoint inhibition. Here, we present Lrp10 as a new negative regulator of CD8 T-cell homeostasis and a host factor that controls tumor resistance with implications for immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Homeostase , Receptores de Interleucina-7 , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos , Receptores de Interleucina-7/metabolismo , Receptores de Interleucina-7/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Transdução de Sinais , Ativação Linfocitária/imunologia , Camundongos Knockout , Camundongos Endogâmicos C57BL , Memória Imunológica , Neoplasias/imunologia , Neoplasias/genética , HumanosRESUMO
Endoplasmic reticulum (ER) calcium (Ca2+ ) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca2+ handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca2+ release from the ER after antigen receptor stimulation. Pacs1-deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1-/- B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1-Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1-Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca2+ handling and presents a new target for lymphoproliferative disease therapy.
Assuntos
Retículo Endoplasmático/metabolismo , Deleção de Genes , Linfopenia/genética , Transtornos Linfoproliferativos/genética , Proteínas Nucleares/genética , Proteínas de Transporte Vesicular/genética , Animais , Linfócitos B/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Linfopenia/metabolismo , Transtornos Linfoproliferativos/metabolismo , Masculino , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Null mutations of spliceosome components or cofactors are homozygous lethal in eukaryotes, but viable hypomorphic mutations provide an opportunity to understand the physiological impact of individual splicing proteins. We describe a viable missense allele (F181I) of Rnps1 encoding an essential regulator of splicing and nonsense-mediated decay (NMD), identified in a mouse genetic screen for altered immune cell development. Homozygous mice displayed a stem cellintrinsic defect in hematopoiesis of all lineages due to excessive apoptosis induced by tumor necrosis factor (TNF)dependent death signaling. Numerous transcript splice variants containing retained introns and skipped exons were detected at elevated frequencies in Rnps1F181I/F181I splenic CD8+ T cells and hematopoietic stem cells (HSCs), but NMD appeared normal. Strikingly, Tnf knockout rescued all hematopoietic cells to normal or near-normal levels in Rnps1F181I/F181I mice and dramatically reduced intron retention in Rnps1F181I/F181I CD8+ T cells and HSCs. Thus, RNPS1 is necessary for accurate splicing, without which disinhibited TNF signaling triggers hematopoietic cell death.
Assuntos
Linfócitos T CD8-Positivos , Ribonucleoproteínas , Animais , Linfócitos T CD8-Positivos/metabolismo , Hematopoese/genética , Homozigoto , Mamíferos/metabolismo , Camundongos , Receptores do Fator de Necrose Tumoral/metabolismo , Ribonucleoproteínas/metabolismo , Deleção de Sequência , Fatores de Necrose Tumoral/metabolismoRESUMO
Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation-phenotype association. At this time, CE has evaluated putative mutation-phenotype associations arising from screening damaging mutations in â¼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.
Assuntos
Mutação em Linhagem Germinativa/genética , Leucócitos/metabolismo , Aprendizado de Máquina , Meiose/genética , Algoritmos , Animais , Automação , Feminino , Citometria de Fluxo , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Probabilidade , Reprodutibilidade dos Testes , SoftwareRESUMO
The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele (xiamen [xm]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3xm displayed a large compensatory alteration in switch I, which adopted a ß-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3xm, strongly associated with and stabilized GPR89, and an N-ethyl-N-nitrosourea (ENU)-induced mutation (explorer) in Gpr89 phenocopied Rabl3xm.
Assuntos
Linfócitos B/imunologia , Linfopoese , Proteínas Mutantes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Linfócitos T/imunologia , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Cristalografia por Raios X , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/imunologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Conformação Proteica , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
PET is a powerful technique for quantifying and visualizing biochemical pathways in vivo. Here, we develop and validate a novel PET probe, [(18)F]-2-deoxy-2-fluoroarabinose ([(18)F]DFA), for in vivo imaging of ribose salvage. DFA mimics ribose in vivo and accumulates in cells following phosphorylation by ribokinase and further metabolism by transketolase. We use [(18)F]DFA to show that ribose preferentially accumulates in the liver, suggesting a striking tissue specificity for ribose metabolism. We demonstrate that solute carrier family 2, member 2 (also known as GLUT2), a glucose transporter expressed in the liver, is one ribose transporter, but we do not know if others exist. [(18)F]DFA accumulation is attenuated in several mouse models of metabolic syndrome, suggesting an association between ribose salvage and glucose and lipid metabolism. These results describe a tool for studying ribose salvage and suggest that plasma ribose is preferentially metabolized in the liver.
Assuntos
Fígado , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacologia , Ribose/metabolismo , Animais , Arabinose/análogos & derivados , Arabinose/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Radioisótopos de Flúor/farmacologia , Glucose/genética , Glucose/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Humanos , Metabolismo dos Lipídeos , Fígado/diagnóstico por imagem , Fígado/metabolismo , Síndrome Metabólica/diagnóstico por imagem , Síndrome Metabólica/metabolismo , Camundongos , Especificidade de Órgãos , RadiografiaRESUMO
Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-ß-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.
Assuntos
Desoxicitidina Quinase/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Animais , Arabinofuranosiluracila/análogos & derivados , Arabinofuranosiluracila/química , Arabinofuranosiluracila/metabolismo , Western Blotting , Linhagem Celular Tumoral , Desoxicitidina Quinase/genética , Feminino , Radioisótopos de Flúor/química , Células-Tronco Hematopoéticas/metabolismo , Imuno-Histoquímica , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Estimativa de Kaplan-Meier , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Endogâmicos , Camundongos Knockout , Camundongos SCID , Mutação , Timo/diagnóstico por imagem , Timo/metabolismo , Fatores de Tempo , Transplante HeterólogoRESUMO
Signals emanating from the T cell receptor (TCR), co-stimulatory receptors, and cytokine receptors each influence CD8 T cell fate. Understanding how these signals respond to homeostatic and microenvironmental cues can reveal new ways to therapeutically direct T cell function. Through forward genetic screening in mice, we discovered that loss-of-function mutations in LDL receptor related protein 10 ( Lrp10 ) caused naïve and central memory CD8 T cells to accumulate in peripheral lymphoid organs. Lrp10 encodes a conserved cell surface protein of unknown immunological function. Lrp10 was induced with T cell activation and its expression post-translationally suppressed IL7 receptor (IL7R) levels. Accordingly, Lrp10 deletion enhanced T cell homeostatic expansion through IL7R signaling. Lrp10 -deficient mice were also intrinsically resistant to syngeneic tumors. This phenotype depended on dense tumor infiltration of CD8 T cells that displayed increased memory cell characteristics, reduced terminal exhaustion, and augmented responses to immune checkpoint inhibition. Here, we present Lrp10 as a new negative regulator of CD8 T cell homeostasis and a host factor that controls tumor resistance with implications for immunotherapy.
RESUMO
A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.
Assuntos
Mucosa Intestinal , Intestinos , Proteínas de Membrana , Animais , Camundongos , Microbioma Gastrointestinal , Glicosilação , Células Caliciformes/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestinos/metabolismo , Proteínas de Membrana/metabolismo , Muco , Celulas de Paneth/metabolismoRESUMO
Non-invasive monitoring of adaptive immunity in infection, cancer, and autoimmunity remains a major challenge. Current techniques to monitor lymphocytes involve numeric and functional determinations of immune cells isolated from the peripheral blood (most often) and tissue (rarely). Invasive measurements are prone to sampling errors and are poorly reflective of the dynamic changes in the location, number, and movement of lymphoid cells. These limitations indicate the need for non-invasive whole-body imaging methodologies that allow longitudinal, quantitative, and functional analyses of the immune system in vivo. Positron emission tomography (PET), a clinically based whole-body imaging modality, has the potential to revolutionize diagnostics and therapeutic monitoring in both clinical and pre-clinical settings. This review discusses studies using PET to image adaptive immune responses in small animal models. We address the challenges inherent in assessing whole-body immunity with PET and recent developments that can improve its performance. Finally, we discuss work to translate PET immune imaging into clinical practice.
Assuntos
Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Tomografia por Emissão de Pósitrons/métodos , Animais , Autoimunidade/imunologia , Diagnóstico por Imagem , Modelos Animais de Doenças , Humanos , Imunidade Ativa/imunologia , Neoplasias/diagnóstico , Neoplasias/imunologiaRESUMO
BACKGROUND & AIMS: Uptake of [18F]1-(2'-deoxy-2'-arabinofuranosyl)cytosine (D-FAC) is a trait of activated lymphocytes; its biodistribution predominates in the spleen, thymus, and bone marrow. In addition, D-FAC is taken up at high levels by the intestine. We analyzed the regional specificity of uptake and cell types that mediate it. METHODS: In mice, 3-dimensional isocontour regions of interest were drawn based on computed tomographic images to quantify intestinal signals from micro-positron emission tomography scans. To ascertain the cell type responsible, intestinal epithelium and immune cells were isolated and D-FAC uptake was analyzed in vitro. Mice deficient in mucosal homing (beta7 integrin-/-), enteric microbiota (germ-free), or active for immune colitis (G alpha i2-/- CD3+ transferred into Rag-/- recipients) were studied. RESULTS: Strong uptake of D-FAC was detected throughout the intestine, with greatest signal per region of interest in the duodenum. Fractionation of intestinal cell types after in vivo uptake revealed that the signal was almost entirely from epithelial cells. Among resident immune cell types, CD4+ T cells showed the greatest per-cell and total uptake. D-FAC uptake increased in both intestinal and systemic lymphoid sites during colitis. Compared with fluorodeoxyglucose, increased uptake of D-FAC in the small and large intestine occurred at an earlier stage of disease development. CONCLUSIONS: Uptake of D-FAC is a prominent trait of normal mouse intestinal epithelial cells, which is useful for their noninvasive visualization by positron emission tomography. Increased uptake of D-FAC reflects the activity of the epithelium and lymphocytes, providing a unique early marker of intestinal inflammation.
Assuntos
Colite/diagnóstico por imagem , Citarabina/análogos & derivados , Mucosa Intestinal/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Animais , Apoptose , Citarabina/farmacocinética , Duodeno/metabolismo , Feminino , Fluordesoxiglucose F18 , Vida Livre de Germes , Mucosa Intestinal/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Tomografia por Emissão de Pósitrons , Distribuição TecidualRESUMO
Adenosine monophosphate deaminase 3 (Ampd3) encodes the erythrocyte isoform of the adenosine monophosphate (AMP) deaminase gene family. Mutations in this gene have been reported in humans, leading to autosomal-recessive erythrocyte AMP deaminase deficiency. However, the mutation is considered clinically asymptomatic. Using N-ethyl-N-nitrosourea mutagenesis to find mutations that affect peripheral lymphocyte populations, we identified 5 Ampd3 mutations (Ampd3guangdong, Ampd3carson, Ampd3penasco, Ampd3taos, and Ampd3commanche) that strongly correlated with a reduction in naive CD4+ T and naive CD8+ T-cell populations. Causation was confirmed by targeted ablation of Ampd3. Knockout mice had reduced frequencies of CD62LhiCD44lo CD4+ naive and CD8+ naive T cells. Interestingly, these phenotypes were restricted to T cells circulating in peripheral blood and were not seen in T cells from secondary lymphoid organs (lymph nodes and spleen). We found that reduction of naive T cells in the peripheral blood of Ampd3-/- mice was caused by T-cell-extrinsic factor(s), which we hypothesize to be elevated levels of adenosine triphosphate released by Ampd3-deficient erythrocytes. These findings provide an example in which disruption of an erythrocyte-specific protein can affect the physiological status of lymphocytes in peripheral blood.
Assuntos
AMP Desaminase , Mutação com Perda de Função , AMP Desaminase/genética , Monofosfato de Adenosina , Animais , Camundongos , Camundongos Knockout , Linfócitos TRESUMO
T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naïve CD4+ T cells to TFH during the adaptive immune response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas Quinases/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Animais , Linfócitos B/imunologia , Transplante de Medula Óssea , Diferenciação Celular , Feminino , Centro Germinativo/imunologia , Células HEK293 , Humanos , Imunoglobulinas/sangue , Imunoterapia Adotiva , Masculino , Camundongos Transgênicos , Mutação , Proteína Quinase D2 , Proteínas Quinases/genéticaRESUMO
Precise control of Wnt signaling is necessary for immune system development. In this study, we detected severely impaired development of all lymphoid lineages in mice, resulting from an N-ethyl-N-nitrosourea-induced mutation in the limb region 1-like gene (Lmbr1l), which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain-containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing "destruction complex" proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/ß-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation, acting as a negative regulator of the Wnt/ß-catenin pathway.
Assuntos
Linfopoese/genética , Receptores de Superfície Celular/fisiologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Superfície Celular/genéticaRESUMO
Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [(18)F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Reparo do DNA/efeitos da radiação , Desoxicitidina Quinase/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Desoxicitidina/metabolismo , Desoxicitidina Quinase/química , Desoxicitidina Quinase/genética , Desoxirribonucleosídeos/metabolismo , Instabilidade Genômica , Hematopoese/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Linfócitos T/citologia , Linfócitos T/fisiologiaRESUMO
Cancer cells and immune cells modulate their metabolism according to specific needs during cancer progression and immune responses. The ability to measure cellular metabolic function in vivo would enable the evaluation of tumors and their response to therapy and also the effectiveness of cellular immune responses to cancer. Positron emission tomography (PET) is a highly sensitive clinical imaging modality that enables whole-body, quantitative measurements of tissue biochemical function. Here, we review work using PET probes for specific metabolic pathways to measure cell function in cancer and immunity. We focus on the use of probes for glycolysis and nucleoside salvage and then discuss the development of new metabolic probes that visualize distinct parameters of cell function during disease.
Assuntos
Fluordesoxiglucose F18 , Imunidade Celular/fisiologia , Neoplasias/diagnóstico , Tomografia por Emissão de Pósitrons/métodos , Proliferação de Células , Humanos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/análiseRESUMO
Clinical tools that measure changes in immune cell metabolism would improve the diagnosis and treatment of immune dysfunction. PET, utilizing probes for specific metabolic processes, detects regions of immune activation in vivo. In this study we investigated the immune cell specificity of PET probes for two different metabolic pathways: [18F]-2-fluorodeoxyglucose ([18F]-FDG) for glycolysis and [18F]-2-fluoro-D-(arabinofuranosyl)cytosine ([18F]-FAC) for deoxycytidine salvage. We isolated innate and adaptive immune cells from tissues of mice challenged with a retrovirus-induced sarcoma and measured their ability to accumulate FDG and FAC. We determined that the two probes had distinct patterns of accumulation: FDG accumulated to the highest levels in innate immune cells, while FAC accumulated predominantly in CD8+ T cells in a manner that correlated with cellular proliferation. This study demonstrates that innate and adaptive cell types differ in glycolytic and deoxycytidine salvage demands during an immune response and that these differential metabolic requirements can be detected with specific PET probes. Our findings have implications for the interpretation of clinical PET scans that use [18F]-FDG or [18F]-FAC to assess immune function in vivo and suggest potential applications of metabolic PET to monitor the effects of targeted immune modulation.
Assuntos
Células/imunologia , Células/metabolismo , Fenômenos do Sistema Imunitário , Redes e Vias Metabólicas , Tomografia por Emissão de Pósitrons/métodos , Animais , Proliferação de Células , Estruturas Celulares , Fluordesoxiglucose F18 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Sensibilidade e EspecificidadeRESUMO
Monitoring immune function with molecular imaging could have a considerable impact on the diagnosis and treatment evaluation of immunological disorders and therapeutic immune responses. Positron emission tomography (PET) is a molecular imaging modality with applications in cancer and other diseases. PET studies of immune function have been limited by a lack of specialized probes. We identified [(18)F]FAC (1-(2'-deoxy-2'-[(18)F]fluoroarabinofuranosyl) cytosine) by differential screening as a new PET probe for the deoxyribonucleotide salvage pathway. [(18)F]FAC enabled visualization of lymphoid organs and was sensitive to localized immune activation in a mouse model of antitumor immunity. [(18)F]FAC microPET also detected early changes in lymphoid mass in systemic autoimmunity and allowed evaluation of immunosuppressive therapy. These data support the use of [(18)F]FAC PET for immune monitoring and suggest a wide range of clinical applications in immune disorders and in certain types of cancer.
Assuntos
Desoxicitidina/análogos & derivados , Radioisótopos de Flúor , Linfocintigrafia , Tomografia por Emissão de Pósitrons/métodos , Cintilografia/métodos , Animais , Desoxicitidina/química , Fluordesoxiglucose F18 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Compostos Radiofarmacêuticos , Sensibilidade e Especificidade , Distribuição TecidualRESUMO
There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats.