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1.
Proc Natl Acad Sci U S A ; 121(14): e2320442121, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38536748

RESUMO

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.


Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Peptídeos/química , Antígenos de Histocompatibilidade/química , Antígenos
2.
Proc Natl Acad Sci U S A ; 120(21): e2221116120, 2023 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-37192158

RESUMO

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.


Assuntos
Neoplasias , Precursores de RNA , Masculino , Humanos , Precursores de RNA/metabolismo , Processamento Alternativo , Leucócitos Mononucleares/metabolismo , Receptores de Antígenos de Linfócitos T , Epitopos de Linfócito T , Imunoterapia , Antígenos de Neoplasias , Peptídeos/metabolismo , Neoplasias/genética , Neoplasias/terapia
3.
Nat Immunol ; 14(10): 1073-83, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24013668

RESUMO

C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.


Assuntos
Transformação Celular Neoplásica/genética , Regulação da Expressão Gênica , Fator de Transcrição Ikaros/genética , Leucemia/genética , Linfopoese/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sequência de Bases , Sítios de Ligação , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Imunoprecipitação da Cromatina , Análise por Conglomerados , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Perfilação da Expressão Gênica , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Fator de Transcrição Ikaros/metabolismo , Imunofenotipagem , Leucemia/metabolismo , Leucemia/mortalidade , Linfoma/genética , Linfoma/metabolismo , Linfoma/mortalidade , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Motivos de Nucleotídeos , Fenótipo , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Timócitos/metabolismo
4.
Proc Natl Acad Sci U S A ; 119(31): e2203410119, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35878026

RESUMO

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.


Assuntos
Fosfatase Ácida , Antígenos de Neoplasias , Receptores de Antígenos de Linfócitos T , Fosfatase Ácida/metabolismo , Antígenos de Neoplasias/metabolismo , Epitopos , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Humanos , Leucócitos Mononucleares , Neoplasias/imunologia , Peptídeos , Receptores de Antígenos de Linfócitos T/metabolismo
5.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33431692

RESUMO

T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 1015 unique sequences. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. Here, we describe the use of mRNA sequencing via cross-linker regulated intracellular phenotype (CLInt-Seq) for efficient recovery of antigen-specific TCRs in cells stained for combinations of intracellular proteins such as cytokines or transcription factors. This method enables high-throughput identification and isolation of low-frequency TCRs specific for any antigen. As a proof of principle, intracellular staining for TNFα and IFNγ identified cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-reactive TCRs with efficiencies similar to state-of-the-art peptide-MHC multimer methodology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. We further optimized the intracellular staining conditions to use a chemically cleavable primary amine cross-linker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ performed similarly to isolation with multimer staining for EBV-reactive TCRs. We anticipate CLInt-Seq will enable droplet-based single-cell mRNA analysis from any tissue where minor populations need to be isolated by intracellular markers.


Assuntos
Fatores de Transcrição Forkhead/genética , Interferon gama/genética , Fator de Necrose Tumoral alfa/genética , Recombinação V(D)J/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Clonagem Molecular , Citomegalovirus/imunologia , Citomegalovirus/patogenicidade , Epitopos/imunologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Humanos , RNA Mensageiro/genética , RNA-Seq , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única , Linfócitos T Reguladores/imunologia , Recombinação V(D)J/imunologia
6.
Proc Natl Acad Sci U S A ; 115(45): E10702-E10711, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30348802

RESUMO

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2-restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.


Assuntos
Proteínas de Homeodomínio/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/isolamento & purificação , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Clonagem Molecular , Humanos
7.
Proc Natl Acad Sci U S A ; 112(5): 1523-8, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25605948

RESUMO

Invariant natural killer T (iNKT) cells comprise a small population of αß T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies, and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited by their small numbers in vivo (∼0.01-1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T-cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. This method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal iNKT cells in mice. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy.


Assuntos
Engenharia Genética , Células-Tronco Hematopoéticas/citologia , Células T Matadoras Naturais/citologia , Transferência Adotiva , Animais , Sequência de Bases , DNA/genética , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/genética , Homologia de Sequência do Ácido Nucleico
8.
Proc Natl Acad Sci U S A ; 110(5): 1857-62, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23319634

RESUMO

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 Heterólogo
9.
bioRxiv ; 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36711524

RESUMO

The ability to selectively bind to antigenic peptides and secrete cytokines can define populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with millions of peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs and secrete cytokines on nanovials, allowing sorting based on both affinity and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes we could link TCR sequence to targets with 100% accuracy. We identified with high specificity an expanded repertoire of functional TCRs targeting viral antigens compared to standard techniques.

10.
Cell Rep ; 37(13): 110167, 2021 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-34919800

RESUMO

Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαß sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαß constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/imunologia , Antígeno HLA-A2/imunologia , SARS-CoV-2/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , COVID-19/terapia , Técnicas de Cultura de Células , Reações Cruzadas/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-A/imunologia , Antígeno HLA-A2/genética , Humanos , Epitopos Imunodominantes/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , RNA Viral/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/imunologia
11.
Mol Cell Biol ; 27(4): 1334-47, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17145776

RESUMO

GPR4 is a G protein-coupled receptor expressed in the vasculature, lung, kidney, and other tissues. In vitro ectopic overexpression studies implicated GPR4 in sensing extracellular pH changes leading to cyclic AMP (cAMP) production. To investigate its biological roles in vivo, we generated GPR4-deficient mice by homologous recombination. Whereas GPR4-null adult mice appeared phenotypically normal, neonates showed a higher frequency of perinatal mortality. The average litter size from GPR4(-/-) intercrosses was approximately 30% smaller than that from GPR4(+/+) intercrosses on N3 and N5 C57BL/6 genetic backgrounds. A fraction of knockout embryos and neonates had spontaneous hemorrhages, dilated and tortuous subcutaneous blood vessels, and defective vascular smooth muscle cell coverage. Mesangial cells in kidney glomeruli were also significantly reduced in GPR4-null neonates. Some neonates exhibited respiratory distress with airway lining cell metaplasia. To examine whether GPR4 is functionally involved in vascular pH sensing, an ex vivo aortic ring assay was used under defined pH conditions. Compared to wild-type aortas, microvessel outgrowth from GPR4-null aortas was less inhibited by acidic extracellular pH. Treatment with an analog of cAMP, a downstream effector of GPR4, abolished microvessel outgrowth bypassing the GPR4-knockout phenotype. These results suggest that GPR4 deficiency leads to partially penetrant vascular abnormalities during development and that this receptor functions in blood vessel pH sensing.


Assuntos
Vasos Sanguíneos/anormalidades , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/metabolismo , Animais , Animais Recém-Nascidos , Aorta/anormalidades , Vasos Sanguíneos/embriologia , Células Cultivadas , Cruzamentos Genéticos , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/patologia , Feminino , Marcação de Genes , Genótipo , Hemorragia/congênito , Humanos , Concentração de Íons de Hidrogênio , Tamanho da Ninhada de Vivíparos , Pulmão/anormalidades , Pulmão/embriologia , Pulmão/patologia , Masculino , Células Mesangiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Penetrância , Doenças Respiratórias/congênito
12.
Proc Natl Acad Sci U S A ; 104(51): 20501-6, 2007 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-18079287

RESUMO

Many cancers and leukemias are associated with strong dominant oncogenic mutations that activate tyrosine kinases and other classes of molecules, including transcription factors and antiapoptotic mechanisms. Some of these events can be targeted with small molecules or antibody-based therapeutics, but many remain intractable. In addition, cancer-related enzyme targets can often mutate, and drug-resistant variants are selected. Therapies directed at the mRNA encoding dominant oncogenes could provide a more global set of technologies for cancer treatment. To test this concept, we have used the model of transformation of hematopoietic cells by the chimeric Bcr-Abl oncogene, a highly activated tyrosine kinase. Our results show that tandem arrays of miRNA mimics, but not single miRNA mimics, directed against the Abl portion of the mRNA and introduced by lentiviral vectors can effectively alter the leukemogenic potency when the degree of suppression of expression of Bcr-Abl is reduced >200-fold from control levels. Only methods capable of such dramatic sustained reduction in the level of expression of highly activated kinase oncogenes are likely to be effective in controlling malignant cell populations.


Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Terapia Genética , Leucemia Linfoide/terapia , MicroRNAs/genética , Animais , Apoptose , Proteínas de Fusão bcr-abl/genética , Lentivirus/genética , Camundongos , Camundongos Endogâmicos
13.
Lab Chip ; 9(4): 555-63, 2009 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-19190791

RESUMO

We have successfully designed and fabricated an integrated microfluidic platform, the hESC-microChip, which is capable of reproducible and quantitative culture and analysis of individual hESC colonies in a semi-automated fashion. In this device, a serpentine microchannel allows pre-screening of dissociated hESC clusters, and six individually addressable cell culture chambers enable parallel hESC culture, as well as multiparameter analyses in sequence. In order to quantitatively monitor hESC proliferation and pluripotency status in real time, knock-in hESC lines with EGFP driven by the endogenous OCT4 promoter were constructed. On-chip immunoassays of several pluripotency markers were carried out to confirm that the hESC colonies maintained their pluripotency. For the first time, our studies demonstrated well characterized hESC culture and analysis in a microfluidic setting, as well as a proof-of-concept demonstration of parallel/multiparameter/real-time/automated examination of self-renewal and differentiation in the same device.


Assuntos
Células-Tronco Embrionárias/citologia , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Cultura de Células , Diferenciação Celular , Processos de Crescimento Celular , Proliferação de Células , Desenho de Equipamento , Humanos , Imuno-Histoquímica , Técnicas Analíticas Microfluídicas/métodos , Fatores de Tempo
14.
Mol Cell Biol ; 26(2): 668-77, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16382156

RESUMO

T-cell death-associated gene 8 (TDAG8) is a G-protein-coupled receptor transcriptionally upregulated by glucocorticoids (GCs) and implicated by overexpression studies in psychosine-mediated inhibition of cytokinesis and in GC-induced apoptosis. To examine the physiological function of TDAG8, we generated knockout (KO) mice by homologous recombination. An enhanced green fluorescent protein reporter was knocked into the disrupted tdag8 locus to allow the analysis of TDAG8 expression in living cells. Interestingly, we found that during thymocyte development, TDAG8 expression resembled the dynamic regulation described for known modulators of GC-induced apoptosis, including Bcl-2, Notch1, and GC receptor. TDAG8 was expressed in double-negative cells, was downregulated at the double-positive transition, and was upregulated in single-positive thymocytes. However, despite this striking expression pattern, maturation and selection of thymocytes, as well as major immune functions, were not affected in TDAG8 KO mice. In contrast to previous overexpression results, TDAG8 was dispensable for psychosine-induced formation of multinucleated cells. Furthermore, TDAG8 KO thymocytes showed normal apoptosis following in vivo and in vitro GC treatment. These results, while establishing a useful reporter strain to study T-lymphocyte maturation, argue against a critical role for TDAG8 in immune development, psychosine-mediated inhibition of cytokinesis, and GC-induced cell death.


Assuntos
Apoptose , Células Gigantes/imunologia , Glucocorticoides/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Linfócitos T/imunologia , Animais , Compartimento Celular , Células Cultivadas , Citocinese/fisiologia , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Ativação Linfocitária , Camundongos , Camundongos Knockout , Psicosina/fisiologia , Receptores Acoplados a Proteínas G/genética , Recombinação Genética
15.
Cell Rep ; 28(10): 2728-2738.e7, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484081

RESUMO

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.


Assuntos
Antígenos de Neoplasias/sangue , Melanoma/sangue , Melanoma/imunologia , Linfócitos T/imunologia , Biópsia , Células HEK293 , Humanos , Imunoterapia , Células Jurkat , Cinética , Linfócitos do Interstício Tumoral/imunologia , Nanopartículas de Magnetita/química , Complexo Principal de Histocompatibilidade , Melanoma/patologia , Melanoma/secundário , Ácidos Nucleicos/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Reprodutibilidade dos Testes , Tomografia Computadorizada por Raios X
16.
Cell Stem Cell ; 25(4): 542-557.e9, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31495780

RESUMO

Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Imunoterapia Adotiva/métodos , Células T Matadoras Naturais/fisiologia , Neoplasias/terapia , Animais , Células Cultivadas , Engenharia Genética , Humanos , Camundongos , Camundongos SCID , Células T Matadoras Naturais/transplante , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Exp Med ; 214(3): 793-814, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28190001

RESUMO

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre-B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre-B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre-B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre-B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre-B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage-specific expression of multiple genes through chromatin compaction at its target genes.


Assuntos
Proteínas de Fusão bcr-abl/análise , Fator de Transcrição Ikaros/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Supressoras de Tumor/fisiologia , Animais , Antígenos CD34/análise , Ciclo Celular , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , Humanos , Fator de Transcrição Ikaros/genética , Leucossialina/análise , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/genética
18.
J Clin Invest ; 125(5): 1815-26, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25822024

RESUMO

Adoptive transfer of tumor-reactive T cells can successfully reduce tumor burden; however, in rare cases, lethal on-target/off-tumor effects have been reported. A noninvasive method to track engineered cells with high sensitivity and resolution would allow observation of correct cell homing and/or identification of dangerous off-target locations in preclinical and clinical applications. Human deoxycytidine kinase triple mutant (hdCK3mut) is a nonimmunogenic PET reporter that was previously shown to be an effective tool to monitor whole-body hematopoiesis. Here, we engineered a construct in which hdCK3mut is coexpressed with the anti-melanoma T cell receptor F5, introduced this construct into human CD34 cells or PBMCs, and evaluated this approach in multiple immunotherapy models. Expression of hdCK3mut allowed engrafted cells to be visualized within recipient bone marrow, while accumulation of [18F]-L-FMAU in hdCK3mut-expressing T cells permitted detection of intratumoral homing. Animals that received T cells coexpressing hdCK3mut and the anti-melanoma T cell receptor had demonstrably higher signals in HLA-matched tumors compared with those in animals that received cells solely expressing hdCK3mut. Engineered T cells caused cytotoxicity in HLA/antigen-matched tumors and induced IFN-γ production and activation. Moreover, hdCK3mut permitted simultaneous monitoring of engraftment and tumor infiltration, without affecting T cell function. Our findings suggest that hdCK3mut reporter imaging can be applied in clinical immunotherapies for whole-body detection of engineered cell locations.


Assuntos
Desoxicitidina Quinase/análise , Genes Reporter , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/terapia , Tomografia por Emissão de Pósitrons , Linfócitos T/imunologia , Animais , Medula Óssea/diagnóstico por imagem , Quimiotaxia de Leucócito , Testes Imunológicos de Citotoxicidade , Desoxicitidina Quinase/genética , Genes Sintéticos , Vetores Genéticos/genética , Sobrevivência de Enxerto , Antígeno HLA-A2/imunologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/fisiologia , Humanos , Imunoterapia Adotiva , Testes de Liberação de Interferon-gama , Lentivirus/genética , Leucócitos Mononucleares/química , Leucócitos Mononucleares/fisiologia , Leucócitos Mononucleares/transplante , Antígeno MART-1/imunologia , Melanoma Experimental/imunologia , Camundongos , Mutação , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Retroviridae/genética , Linfócitos T/transplante , Timo/transplante
19.
PLoS One ; 9(8): e104125, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25101980

RESUMO

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/fisiologia
20.
Blood ; 110(6): 1831-9, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17554060

RESUMO

Reduced lymphopoiesis during aging contributes to declines in immunity, but little consideration has been given to its effect on the development of hematologic disease. This report demonstrates that age-related defects in lymphopoiesis underlie the myeloid dominance of adult leukemia. Using a murine model of chronic myeloid leukemia, an adult-onset malignancy that arises from transformation of hematopoietic stem cells by the BCR-ABL(P210) oncogene, we demonstrate that young bone marrow (BM) cells that were transformed with BCR-ABL(P210) initiated both a myeloproliferative disorder (MPD) and B-lymphoid leukemia, whereas BCR-ABL(P210)-transformed old BM cells recapitulated the human disease by inducing an MPD with rare lymphoid involvement. In addition, the lesser severity of MPDs initiated from old BCR-ABL(P210)-transduced BM cells revealed unappreciated defects in aged myeloid progenitors. These data demonstrate that aging affects patterns of leukemogenesis and indicate that the effects of senescence on hematopoiesis are more extensive than previously appreciated.


Assuntos
Envelhecimento , Linfócitos B/patologia , Células-Tronco Hematopoéticas/fisiologia , Leucemia Linfoide/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/etiologia , Linfopoese , Células Mieloides/patologia , Adulto , Idade de Início , Animais , Transplante de Medula Óssea , Modelos Animais de Doenças , Citometria de Fluxo , Proteínas de Fusão bcr-abl/fisiologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Transtornos Mieloproliferativos/patologia , Retroviridae
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