a-simple-rapid-protocol-to-non-enzymatically-dissociate-fresh-human

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Université Libre de Bruxelles

Human immunodeficiency virus, type 1 (HIV-1) infection of CD4(+) T cells progressively abrogates T cell receptor (TCR).CD3 function and surface expression by specifically interfering with CD3gamma gene transcription. Our data show that the loss of CD3gamma transcripts begins very early after infection and accumulates to a >90% deficiency before a significant effect on surface receptor density is apparent. Blocking TCR.CD3-directed NFAT activation with cyclosporin A provokes a partial re-expression of CD3gamma gene transcripts and surface complexes in a time- and dose-dependent manner. We have identified three NFAT consensus sequences (5'-GGAAA-3') in the 5'-upstream region of the human CD3gamma gene at: -124 to -120 (NFAT(gamma1)), -384 to -380 (NFAT(gamma2)), and +450 to +454 (NFAT(gamma3)) from the first transcription initiation site. Using electrophoretic mobility shift and supershift assays, we show that NFATc2 alone binds to the NFAT(gamma2) motif; however, complexes containing either NFATc2 or NFATc1 plus NF-kappaB p50 bind to the NFAT(gamma1) and NFAT(gamma3) sites. We further demonstrate that NFATc1 and NF-kappaB p50 bind in the same protein.DNA complex and that a fourth Ala added to the core sequence (5'-GGAAAA-3') in NFAT(gamma1), and NFAT(gamma3) is critical for their binding. Finally, we have shown that an increase in the binding of nuclear NFATc2, NFATc1, and NF-kappaB p50 to these three motifs is correlated with a progressive loss of CD3gamma transcripts after HIV-1 infection.

Identification of three NFAT binding motifs in the 5'-upstream region of the human CD3gamma gene that differentially bind NFATc1, NFATc2, and NF-kappa B p50.

Growing evidence that the CD3gamma gene is specifically targeted in some T cell diseases focused our attention on the need to identify and characterize the elusive elements involved in CD3gamma transcriptional control. In this study, we show that while the human CD3gamma and CD3delta genes are oriented head-to-head and separated by only 1.6 kb, the CD3gamma gene is transcribed from an independent but weak, lymphoid-specific TATA-less proximal promoter. Using RNA ligase-mediated rapid amplification of cDNA ends, we demonstrate that a cluster of transcription initiation sites is present in the vicinity of the primary core promoter, and the major start site is situated in a classical initiator sequence. A GT box immediately upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these adjacent elements enhanced by the presence of a second GC box 10 nt further upstream. The primary core promoter is limited to a sequence that extends upstream to -15 and contains the initiator and GT box. An identical GT box located approximately 50 nt from the initiator functions as a weak secondary core promoter and likely generates transcripts originating upstream from the +1. Finally, we show that two previously identified NFAT motifs in the proximal promoter positively (NFATgamma(1)) or negatively (NFATgamma(1) and NFATgamma(2)) regulate expression of the human CD3gamma gene by their differential binding of NFATc1 plus NF-kappaB p50 or NFATc2 containing complexes, respectively. These data elucidate some of the mechanisms controlling expression of the CD3gamma gene as a step toward furthering our understanding of how its transcription is targeted in human disease.

Transcriptional regulation of the human CD3 gamma gene: the TATA-less CD3 gamma promoter functions via an initiator and contiguous Sp-binding elements.

The lymphocytic variant of hypereosinophilic syndrome (LV-HES) is an underrated disease defined by the monoclonal proliferation of interleukin-5 secreting T-cells. This disease is distinguished by a period of chronic lymphoproliferation without clinical transformation, which is frequently a precursor to T-cell lymphoma. In this study, LV-HES was used as a model of pre-malignancy to identify specific marker(s) predictive of the potential for malignant transformation.

6q- is an early and persistent chromosomal aberration in CD3-CD4+ T-cell clones associated with the lymphocytic variant of hypereosinophilic syndrome.

Determine the molecular defects underlying the CD3(-)CD4(+) T-cell phenotype and persistence of this clonal population in patients with hypereosinophilic syndrome.

Defective CD3gamma gene transcription is associated with NFATc2 overexpression in the lymphocytic variant of hypereosinophilic syndrome.

Pertussis toxin (PTX) is known to be mitogenic for T lymphocytes, but its direct action on naive human T cells has not been specified. Herein, we show that PTX induces the proliferation of purified adult CD45RA(+)CD4(+) T cells independently of its ADP-ribosyltransferase activity. PTX directly induces TNF-alpha and IL-2 mRNA expression, modulates the level of several cell surface receptors and induces Forkhead box p3 (Foxp3) protein accumulation in naive CD4(+) T cells. Addition of autologous dendritic cells was found to be required for the production of high levels of IFN-gamma by PTX-stimulated naive T cells. These effects of PTX occurred in conjunction with activation of NF-kappaB and NFAT transcription factors. Overall, responses of neonatal CD4(+) T cells to PTX were similar to those of adult CD45RA(+)CD4(+) naive T cells except for their blunted CD40 ligand up-regulation. We suggest that the adjuvant properties of PTX during primary cell-mediated immune responses involve a direct action on naive T lymphocytes in addition to activation of antigen-presenting cells.

Pertussis toxin activates adult and neonatal naive human CD4+ T lymphocytes.

Preclinical and clinical studies have shown that the proteasome inhibitor bortezomib (PS341, Velcade) is highly effective when combined with chemotherapeutic agents. The value of trastuzumab (Herceptin) in HER-2-positive (3+ score by immunohistochemistry or fluorescence in situ hybridization positive) breast cancer is also known; however, the response rate is <40% for metastatic breast cancer. These two pharmacologic agents prevent nuclear factor-kappaB (NF-kappaB) activation and induce nuclear accumulation of the cyclin-dependent kinase inhibitor p27(kip1), suggesting that combining bortezomib with trastuzumab could increase trastuzumab efficacy.

Bortezomib (PS-341, Velcade) increases the efficacy of trastuzumab (Herceptin) in HER-2-positive breast cancer cells in a synergistic manner.

HTLV-I infected CD4+ T-cells lines usually progress towards a CD3- or CD3low phenotype. In this paper, we studied expression, kinetics, chromatin remodeling of the CD3 gene at different time-points post HTLV-I infection.

Progressive loss of CD3 expression after HTLV-I infection results from chromatin remodeling affecting all the CD3 genes and persists despite early viral genes silencing.

The clonal CD3(-)CD4(+) T-cell population characterizing lymphocytic variant hypereosinophilic syndrome (L-HES) persists for years, with a subgroup of patients ultimately progressing to T lymphoma. The molecular changes associated with the premalignant clone and the emergence of malignant subclones are unknown, precluding the development of targeted therapy for this HES variant. In this study, we used whole genome arrays to examine gene expression in the CD3(-)CD4(+) T cells and found that 850 genes were differentially regulated during chronic disease compared with CD3(+)CD4(+) T cells from healthy donors. Changes in the expression of 349 genes were altered in association with the clinical progression from chronic L-HES to T lymphoma in 1 patient, with 87 of 349 genes representing further changes in genes whose expression was altered in all chronic disease patients (87 of 850). Array analysis after CD2/CD28-mediated activation revealed that the major gene expression changes observed in the CD3(-)CD4(+) T cells do not reflect activation induced alterations but rather pathways involved in T-cell homeostasis, including transforming growth factor-beta signaling, apoptosis, and T-cell maturation, signaling, and migration. Examination of microRNA expression in the CD3(-)CD4(+) T cells from patients with chronic disease identified 23 microRNAs that changed significantly, among which miR-125a further decreased in association with one patient's evolution to T lymphoma.

Molecular profiling of CD3-CD4+ T cells from patients with the lymphocytic variant of hypereosinophilic syndrome reveals targeting of growth control pathways.

&#x6162;&#x75C5;&#x6BD2;&#x8F7D;&#x4F53; (LV) &#x662F;&#x4E3B;&#x7BA1;&#x57FA;&#x56E0;&#x8F6C;&#x79FB;&#x8F66;&#x8F86;&#xFF0C;&#x7528;&#x4E8E;&#x7814;&#x7A76;&#x548C;&#x57FA;&#x56E0;&#x7597;&#x6CD5;&#x7684;&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#xFF0C;&#x7531;&#x4E8E;&#x5176;&#x7A33;&#x5B9A;&#x548C;&#x4E00;&#x4F53;&#x5316;&#x7684;&#x975E;&#x5212;&#x5206;&#x5212;&#x5206;&#x5355;&#x5143;&#x683C;&#x548C;&#x957F;&#x671F;&#x7684;&#x8F6C;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x3002;&#x6211;&#x4EEC; LV &#x7684;&#x7406;&#x89E3;&#x4EE5;&#x53CA;&#x63D0;&#x4F9B;&#x89E3;&#x51B3;&#x65B9;&#x6848;&#x7684;&#x57FA;&#x56E0;&#x6CBB;&#x7597;&#xFF0C;&#x5173;&#x6CE8;&#x751F;&#x7269;&#x5B89;&#x5168;&#x95EE;&#x9898;&#x5DF2;&#x7ECF;&#x53D1;&#x73B0;&#x63D2;&#x5165;&#x7A81;&#x53D8;&#x7684;&#x98CE;&#x9669;&#x7684;&#x590D;&#x5236;&#x4E3B;&#x7BA1;&#x6162; (RCL) &#x4EE3;&#x5E76;&#x77E2;&#x91CF;&#x8C03;&#x52A8;&#x3002;&#x7814;&#x7A76;&#x8005;&#x56E0;&#x6B64;&#x7EE7;&#x7EED;&#x505A;&#x51FA;&#x91CD;&#x5927;&#x52AA;&#x529B;&#x5728; LV &#x8BBE;&#x8BA1;&#x4E0E;&#x6539;&#x8FDB;&#x7684;&#x6548;&#x679C;&#x548C;&#x751F;&#x7269;&#x5B89;&#x5168;&#x529F;&#x80FD;&#x3002;&#x5B9E;&#x9A8C;&#x7814;&#x7A76;&#x7279;&#x522B;&#x662F; LV &#x7CFB;&#x7EDF;&#x7684;&#x9009;&#x62E9;&#x5F80;&#x5F80;&#x662F;&#x7531;&#x529F;&#x80FD;&#x7684;&#x8003;&#x8651;&#xFF0C;&#x5305;&#x62EC;&#x63D0;&#x9AD8;&#x7684;&#x751F;&#x4EA7;&#x529B;&#x548C;/&#x6216;&#x8F6C;&#x5BFC;&#x6548;&#x7387;&#x9A71;&#x52A8;&#x7684;&#x3002;&#x66F4;&#x5B89;&#x5168;&#x7684;&#x8F7D;&#x4F53;&#x7684;&#x8BBE;&#x8BA1;&#x5DF2;&#x8FD8;&#x76F4;&#x63A5;&#x53D7;&#x60E0;&#xFF0C;&#x7814;&#x7A76;&#x4EBA;&#x5458;&#x8BA9;&#x4ED6;&#x4EEC;&#x8FDB;&#x884C;&#x98CE;&#x9669;&#x8F83;&#x4F4E;&#x7684;&#x5B9E;&#x9A8C;&#x7814;&#x7A76;&#x3002;&#x76EE;&#x524D;&#xFF0C;&#x8F7D;&#x4F53;&#x7ED3;&#x5408;&#x589E;&#x52A0;&#x7684;&#x8F6C;&#x5BFC;&#x6548;&#x7387;&#x6539;&#x8FDB;&#x7684;&#x5B89;&#x5168;&#x529F;&#x80FD; &#xFF08;&#x5373;&#x51CF;&#x5C11;&#x91CD;&#x7EC4;&#x548C;&#x77E2;&#x91CF;&#x8C03;&#x52A8;&#x7684;&#x98CE;&#x9669;)&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x98CE;&#x9669;&#x4E0E;&#x7EC6;&#x80DE;&#x5728;&#x8BC4;&#x4F30;&#x8FD9;&#x4E9B;&#x5A92;&#x4ECB;&#x7684;&#x6574;&#x4F53;&#x98CE;&#x9669;&#x7684;&#x8C03;&#x67E5;&#x5458;&#x589E;&#x76CA;&#x66F4;&#x5927;&#x91CD;&#x8981;&#x6027;&#x7684;&#x65E0;&#x610F;&#x4E2D;&#x8F6C;&#x5BFC;&#x76F8;&#x5173;&#x8054;&#xFF0C;&#x6210;&#x4E3A;&#x91CD;&#x8981;&#x7684;&#x751F;&#x7269;&#x5B89;&#x5168;&#x95EE;&#x9898;&#x3002;&#x8FD9;&#x4E00;&#x5BA1;&#x67E5;&#x6982;&#x8FF0;&#x4E86;&#x7528;&#x6765;&#x6539;&#x5584; LV &#x751F;&#x7269;&#x5B89;&#x5168;&#x7684;&#x6BD4;&#x8F83;&#x5148;&#x8FDB;&#x7684;&#x548C;&#x65B0;&#x51FA;&#x73B0;&#x7684; LV &#x751F;&#x4EA7;&#x7CFB;&#x7EDF;&#x548C;&#x7A81;&#x51FA;&#x5176;&#x6240;&#x5305;&#x542B;&#x7684;&#x4F7F;&#x7528;&#x8FC7;&#x7A0B;&#x4E2D;&#x53EF;&#x80FD;&#x51FA;&#x73B0;&#x7684;&#x751F;&#x7269;&#x5B89;&#x5168;&#x95EE;&#x9898;&#x7684;&#x4E0D;&#x540C;&#x7B56;&#x7565;&#x3002;&#x4E13;&#x95E8;&#x89E3;&#x51B3; LV &#x5728;&#x7814;&#x7A76;&#x4E2D;&#x7684;&#x4F7F;&#x7528;&#x7684;&#x51E0;&#x4E2A;&#x73B0;&#x6709;&#x56FD;&#x5BB6;&#x548C;&#x56FD;&#x9645;&#x751F;&#x7269;&#x5B89;&#x5168;&#x5EFA;&#x8BAE;&#x8FDB;&#x884C;&#x8BA8;&#x8BBA;&#x548C;&#x5EFA;&#x8BAE;&#x4F7F;&#x7528; LV &#x7684;&#x6700;&#x5E38;&#x89C1;&#x7684;&#x7814;&#x7A76;&#x6D3B;&#x52A8;&#x3002;

&#x5148;&#x8FDB;&#x7684;&#x6162;&#x75C5;&#x6BD2;&#x8F7D;&#x4F53;&#x7684;&#x7814;&#x7A76;&#x4F7F;&#x7528;&#xFF1A; &#x98CE;&#x9669;&#x8BC4;&#x4F30;&#x548C;&#x751F;&#x7269;&#x5B89;&#x5168;&#x7684;&#x5EFA;&#x8BAE;&#x3002;

Lentiviral vectors (LV) are competent gene transfer vehicles, as used for both research and gene therapy applications, because of their stable integration in non-dividing and dividing cells and long-term transgene expression. Along with our understanding that LV offer solutions for gene therapy, biosafety concerns have uncovered risks due to insertional mutagenesis, the generation of replication competent lentiviruses (RCL) and vector mobilization. Researchers therefore continue to devote significant efforts in designing LV with improved efficacy and biosafety features. The choice of a particular LV system for experimental studies is often driven by functional considerations, including increased productivity and/or transduction efficiency. The design of safer vectors has also directly benefited researchers allowing them to conduct experimental studies with lower risk. Currently, vectors combine improved safety features (that decrease the risk of recombination and vector mobilization) with increased transduction efficiency. Hence, risks associated with the inadvertent transduction of cells of the investigator gain greater importance in assessing the overall risk of these vectors and become an important biosafety concern. This review outlines the different strategies used to improve LV biosafety by comparing state-of-the-art and emerging LV production systems and highlighting biosafety issues that can arise during their contained use. The few existing national and international biosafety recommendations that specifically address the use of LV in research are discussed and recommendations for most common research activities using LV are proposed.

State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations.

Vecteurs LENTIVIRAUX (LV) sont des v&eacute;hicules de transfert de g&egrave;ne comp&eacute;tent, tel qu&#39;utilis&eacute; pour les applications de la recherche et le g&egrave;ne th&eacute;rapie, en raison de leur int&eacute;gration stable en non-divisant et en divisant les cellules et l&#39;expression de transg&egrave;nes &agrave; long terme. Ainsi que notre compr&eacute;hension que LV offrent des solutions pour la th&eacute;rapie g&eacute;nique, les pr&eacute;occupations de la pr&eacute;vention des risques biotechnologiques ont d&eacute;couvert des risques dus &agrave; la mutagen&egrave;se insertionnelle, la g&eacute;n&eacute;ration de lentivirus comp&eacute;tente de r&eacute;plication (RCL) et vecteur de mobilisation. Par cons&eacute;quent, les chercheurs continuent de consacrer des efforts consid&eacute;rables dans la conception de LV avec efficacit&eacute; am&eacute;lior&eacute;e et des fonctionnalit&eacute;s de pr&eacute;vention des risques biotechnologiques. Le choix d&#39;un syst&egrave;me particulier de LV dans les &eacute;tudes exp&eacute;rimentales est souvent motiv&eacute; par des consid&eacute;rations fonctionnelles, y compris accro&icirc;tre l&#39;efficience de la productivit&eacute; et/ou de transduction. La conception de vecteurs plus s&ucirc;res a &eacute;galement directement b&eacute;n&eacute;fici&eacute; chercheurs leur permettant, pour mener des &eacute;tudes exp&eacute;rimentales avec moins de risques. Actuellement, vecteurs de combinent les caract&eacute;ristiques de s&eacute;curit&eacute; am&eacute;lior&eacute;es (qui diminuent le risque de recombinaison et de mobilisation du vecteur) avec une efficacit&eacute; accrue de la transduction. Donc, risques associ&eacute;s &agrave; la transduction par inadvertance des cellules de l&#39;enqu&ecirc;teur gain plus d&#39;importance dans l&#39;&eacute;valuation du risque global de ces vecteurs et deviennent une pr&eacute;occupation importante de bios&eacute;curit&eacute;. Cette revue d&eacute;crit les diff&eacute;rentes strat&eacute;gies utilis&eacute;es pour am&eacute;liorer la bios&eacute;curit&eacute; LV en comparant l&#39;&eacute;tat-of-the-art et nouveaux syst&egrave;mes de production de LV et mettant en lumi&egrave;re les questions de bios&eacute;curit&eacute; qui peuvent survenir au cours de leur utilisation confin&eacute;e. Quelques recommandations nationales et internationales sur la bios&eacute;curit&eacute; existantes qui visent express&eacute;ment &agrave; l&#39;utilisation de LV en recherche sont discut&eacute;es et recommandations pour les activit&eacute;s de recherche plus courantes &agrave; l&#39;aide de LV sont propos&eacute;es.

Vecteurs LENTIVIRAUX &eacute;tat-of-the-art pour la recherche : recommandations de pr&eacute;vention des risques biotechnologiques et de l&#39;&eacute;valuation des risques.

Lentivirale Vektoren (LV) sind zust&auml;ndige gen Transfer Fahrzeuge, f&uuml;r Forschung und gen-Therapie-Anwendungen aufgrund ihrer stabilen Integration in nicht teilenden und Teilen von Zellen und langfristige Transgenexpression verwendet. Zusammen mit unserem Verst&auml;ndnis, die LV bieten L&ouml;sungen f&uuml;r Gentherapie, biologische Sicherheit Bedenken haben Risiken durch muss Mutagenese, entdeckt die Generation der Replikation zust&auml;ndigen Lentiviruses (RCL) und Vektor-Mobilisierung. Forscher daher weiterhin erhebliche Anstrengungen bei der Gestaltung von LV mit verbesserter Wirksamkeit und biologische Funktionen widmen. Die Wahl eines bestimmten LV-Systems f&uuml;r experimentelle Studien wird oft mit funktionalen &Uuml;berlegungen, einschlie&szlig;lich erh&ouml;hte Produktivit&auml;t bzw. Transduktion Effizienz angetrieben. Das Design der sicherer Vektoren profitierte auch direkt Forscher Ihnen um experimentelle Studien bei geringerem Risiko. Derzeit kombinieren Vektoren verbesserte Sicherheits-Features (das verringern das Risiko der Rekombination und Vektor-Mobilisierung) mit erh&ouml;hter Transduktion Effizienz. Daher Risiken der unbeabsichtigten Transduktion von Zellen der Ermittler Gewinn gr&ouml;&szlig;ere Bedeutung bei der Beurteilung des Gesamtrisikos dieser Vektoren zugeordnet und eine wichtige biologische Sicherheit Anliegen geworden. Diesen Bericht werden die verschiedenen Strategien zur Verbesserung der LV biologische Sicherheit durch den Vergleich Staat-of-the-Art und aufstrebenden LV-Produktionssysteme und biologische Sicherheit Fragen, die w&auml;hrend ihrer Anwendung in geschlossenen Systemen auftreten k&ouml;nnen. Die paar vorhandenen nationalen und internationalen Biosafety-Empfehlungen, die speziell den Einsatz von LV in der Forschung befasst werden besprochen und Empfehlungen f&uuml;r die h&auml;ufigsten Forschungsaktivit&auml;ten mit LV werden vorgeschlagen.

Zustand-of-the-Art Lentiviralen Vektoren f&uuml;r Forschungszwecke: risk Assessment und biologische Sicherheit Empfehlungen.

Vettori lentivirali (LV) sono veicoli di trasferimento del gene competente, come usato per applicazioni di ricerca e di gene terapia, a causa della loro integrazione stabile in non-dividendo e dividendo le cellule e a lungo termine espressione del transgene. Insieme con la nostra comprensione che LV offrono soluzioni per la terapia genica, biosicurezza preoccupazioni hanno scoperto i rischi dovuti alla mutagenesi inserzionale, la generazione di lentiviruses competente replica (RCL) e vector mobilitazione. I ricercatori pertanto continuano a dedicare sforzi significativi nella progettazione di LV con maggiore efficacia e funzionalit&agrave; di biosicurezza. La scelta di un particolare sistema di LV per studi sperimentali &egrave; spesso guidata da considerazioni funzionali, tra cui efficienza aumentata produttivit&agrave; e/o trasduzione. La progettazione di vettori pi&ugrave; sicuri ha beneficiato direttamente anche ricercatori permettendo loro di condurre studi sperimentali con minor rischio. Attualmente, vettori combinano caratteristiche di miglioramento della sicurezza (che riducono il rischio di ricombinazione e mobilitazione vettoriale) con efficienza aumentata trasduzione. Quindi, i rischi associati con la trasduzione accidentale delle cellule dell&#39;importanza maggiore investigatore guadagno nel valutare il rischio complessivo di questi vettori e diventano una preoccupazione importante sulla biosicurezza. Questa recensione delinea le diverse strategie permette migliorare LV biosicurezza confrontando stato-of-the-art ed emergenti sistemi di produzione di LV e mettendo in evidenza problemi di biosicurezza che possono verificarsi durante il loro impiego confinato. Le poche raccomandazioni biosicurezza nazionali ed internazionali esistenti che affrontano in modo specifico l&#39;uso di LV nella ricerca sono discussi e raccomandazioni per le attivit&agrave; di ricerca pi&ugrave; comuni utilizzando LV sono proposti.

Stato-of-the-art vettori lentivirali per uso di ricerca: valutazione e biosicurezza raccomandazioni del rischio.

&#x30EC;&#x30F3;&#x30C1;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x30D9;&#x30AF;&#x30BF;&#x30FC; (LV) &#x3068;&#x306F;&#x3001;&#x7814;&#x7A76;&#x3068;&#x907A;&#x4F1D;&#x5B50;&#x7642;&#x6CD5;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3067;&#x306F;&#x3001;&#x975E;&#x5206;&#x88C2;&#x7D30;&#x80DE;&#x3068;&#x9577;&#x671F;&#x7684;&#x306A;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x3092;&#x5206;&#x5272;&#x3067;&#x306E;&#x5B89;&#x5B9A;&#x3057;&#x305F;&#x7D71;&#x5408;&#x306E;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x4E3B;&#x52D9;&#x907A;&#x4F1D;&#x5B50;&#x4F1D;&#x9054;&#x8ECA;&#x4E21;&#x3092;&#x3068;&#x304A;&#x308A;&#x3002;LV &#x306E;&#x6211;&#x3005; &#x306E;&#x7406;&#x89E3;&#x3068;&#x5171;&#x306B;&#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x63D0;&#x4F9B;&#x907A;&#x4F1D;&#x5B50;&#x6CBB;&#x7642;&#x3001;&#x30D0;&#x30A4;&#x30AA; &#x30BB;&#x30FC;&#x30D5;&#x30C6;&#x30A3;&#x306E;&#x61F8;&#x5FF5;&#x5206;&#x5B50;&#x6A19;&#x7684;&#x7A81;&#x7136;&#x5909;&#x7570;&#x8A98;&#x767A;&#x306B;&#x3088;&#x308B;&#x30EA;&#x30B9;&#x30AF;&#x304C;&#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x4E3B;&#x52D9;&#x30D7;&#x30ED;&#x30C8;&#x30BF;&#x30A4;&#x30D7;&#x30EC; (RCL) &#x306E;&#x751F;&#x6210;&#x3092;&#x767A;&#x898B;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x52D5;&#x54E1;&#x3002;&#x7814;&#x7A76;&#x8005;&#x306F;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066; LV &#x306E;&#x6539;&#x5584;&#x52B9;&#x679C;&#x3068;&#x30D0;&#x30A4;&#x30AA; &#x30BB;&#x30FC;&#x30D5;&#x30C6;&#x30A3;&#x6A5F;&#x80FD;&#x3092;&#x5099;&#x3048;&#x305F;&#x8A2D;&#x8A08;&#x306B;&#x591A;&#x5927;&#x306A;&#x52AA;&#x529B;&#x3092;&#x50BE;&#x6CE8;&#x3057;&#x7D9A;&#x3051;&#x307E;&#x3059;&#x3002;&#x7279;&#x5B9A;&#x306E; LV &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x5B9F;&#x9A13;&#x7814;&#x7A76;&#x306E;&#x305F;&#x3081;&#x306E;&#x9078;&#x629E;&#x306F;&#x3001;&#x3057;&#x3070;&#x3057;&#x3070;&#x751F;&#x7523;&#x6027;&#x304A;&#x3088;&#x3073;/&#x307E;&#x305F;&#x306F;&#x4F1D;&#x9054;&#x52B9;&#x7387;&#x306E;&#x5411;&#x4E0A;&#x3092;&#x542B;&#x3080;&#x3001;&#x6A5F;&#x80FD;&#x306E;&#x8003;&#x616E;&#x4E8B;&#x9805;&#x306B;&#x3088;&#x3063;&#x3066;&#x99C6;&#x52D5;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x5B89;&#x5168;&#x306A;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x306E;&#x30C7;&#x30B6;&#x30A4;&#x30F3;&#x306F;&#x3088;&#x3046;&#x306B;&#x305D;&#x308C;&#x3089;&#x306E;&#x4F4E;&#x3044;&#x30EA;&#x30B9;&#x30AF;&#x3067;&#x306E;&#x5B9F;&#x8A3C;&#x7814;&#x7A76;&#x306E;&#x7814;&#x7A76;&#x8005;&#x3082;&#x76F4;&#x63A5;&#x6069;&#x6075;&#x3092;&#x53D7;&#x3051;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x73FE;&#x5728;&#x3001;&#x30D9;&#x30AF;&#x30C8;&#x30EB; (&#x7D44;&#x63DB;&#x3048;&#x3068;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x52D5;&#x54E1;&#x306E;&#x30EA;&#x30B9;&#x30AF;&#x3092;&#x6E1B;&#x5C11;&#x3055;&#x305B;&#x308B;) &#x306E;&#x6539;&#x826F;&#x3055;&#x308C;&#x305F;&#x5B89;&#x5168;&#x6A5F;&#x80FD;&#x5897;&#x52A0;&#x4F1D;&#x9054;&#x52B9;&#x7387;&#x3068;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x30EA;&#x30B9;&#x30AF;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3053;&#x308C;&#x3089;&#x306E;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x306E;&#x5168;&#x4F53;&#x306E;&#x30EA;&#x30B9;&#x30AF;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x8ABF;&#x67FB;&#x5B98;&#x5229;&#x5F97;&#x306E;&#x5927;&#x304D;&#x3044;&#x91CD;&#x8981;&#x6027;&#x7D30;&#x80DE;&#x306E;&#x4E0D;&#x6CE8;&#x610F;&#x306B;&#x3088;&#x308B;&#x60C5;&#x5831;&#x4F1D;&#x9054;&#x3068;&#x30D0;&#x30A4;&#x30AA; &#x30BB;&#x30FC;&#x30D5;&#x30C6;&#x30A3;&#x306E;&#x91CD;&#x8981;&#x306A;&#x95A2;&#x5FC3;&#x4E8B;&#x3068;&#x306A;&#x3063;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30EC;&#x30D3;&#x30E5;&#x30FC;&#x306F; LV &#x30D0;&#x30A4;&#x30AA;&#x30BB;&#x30A4;&#x30D5;&#x30C6;&#x30A3; &#x30A2;&#x30FC;&#x30C8;&#x306E;&#x72B6;&#x614B;&#x3092;&#x6BD4;&#x8F03;&#x3057;&#x3066; LV &#x751F;&#x7523;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x65B0;&#x8208;&#x306E;&#x542B;&#x307E;&#x308C;&#x3066;&#x3044;&#x308B;&#x4F7F;&#x7528;&#x4E2D;&#x306B;&#x8D77;&#x3053;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x30D0;&#x30A4;&#x30AA; &#x30BB;&#x30FC;&#x30D5;&#x30C6;&#x30A3;&#x306E;&#x554F;&#x984C;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x3066;&#x6539;&#x5584;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x5225;&#x306E;&#x6226;&#x7565;&#x3092;&#x6982;&#x8AAC;&#x3057;&#x307E;&#x3059;&#x3002;&#x7279;&#x306B; LV &#x7814;&#x7A76;&#x3067;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x30A2;&#x30C9;&#x30EC;&#x30B9;&#x3044;&#x304F;&#x3064;&#x304B;&#x65E2;&#x5B58;&#x306E;&#x56FD;&#x5185;&#x30FB;&#x56FD;&#x969B;&#x30D0;&#x30A4;&#x30AA; &#x30BB;&#x30FC;&#x30D5;&#x30C6;&#x30A3;&#x63D0;&#x8A00;&#x304C;&#x8B70;&#x8AD6;&#x3055;&#x308C;&#x3001;LV &#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x6700;&#x3082;&#x4E00;&#x822C;&#x7684;&#x306A;&#x7814;&#x7A76;&#x6D3B;&#x52D5;&#x306E;&#x305F;&#x3081;&#x306E;&#x63A8;&#x5968;&#x4E8B;&#x9805;&#x3092;&#x63D0;&#x6848;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x72B6;&#x614B;&#x306E;&#x82B8;&#x8853;&#x30EC;&#x30F3;&#x30C1;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x30D9;&#x30AF;&#x30BF;&#x30FC;&#x7814;&#x7A76;&#x7528;&#xFF1A; &#x30EA;&#x30B9;&#x30AF; &#x30A2;&#x30BB;&#x30B9;&#x30E1;&#x30F3;&#x30C8;&#x3068;&#x30D0;&#x30A4;&#x30AA; &#x30BB;&#x30FC;&#x30D5;&#x30C6;&#x30A3;&#x306B;&#x95A2;&#x3059;&#x308B;&#x63A8;&#x5968;&#x4E8B;&#x9805;&#x3002;

Lentiviral &#xBCA1;&#xD130; (LV)&#xB294; &#xC720;&#xB2A5;&#xD55C; &#xC9C4; &#xC774;&#xB3D9; &#xCC28;&#xB7C9; &#xBE44; &#xBD84;&#xD560; &#xD558; &#xACE0; &#xBD84;&#xD560; &#xC138;&#xD3EC; &#xBC0F; &#xC7A5;&#xAE30; transgene &#xC2DD;&#xC5D0;&#xC11C;&#xC758; &#xC548;&#xC815;&#xC801;&#xC778; &#xD1B5;&#xD569; &#xB54C;&#xBB38;&#xC5D0; &#xC5F0;&#xAD6C;&#xC640; &#xC720;&#xC804;&#xC790; &#xCE58;&#xB8CC; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC5D0; &#xC0AC;&#xC6A9; &#xB418;&#xB294;. &#xC194;&#xB8E8;&#xC158;&#xC744; &#xC81C;&#xACF5; &#xD558;&#xB294; LV &#xC6B0;&#xB9AC;&#xC758; &#xC774;&#xD574; &#xD568;&#xAED8; &#xC720;&#xC804;&#xC790; &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; biosafety &#xC6B0;&#xB824; insertional mutagenesis &#xC778;&#xD574; &#xC704;&#xD5D8; &#xBCF5;&#xC81C; &#xC720;&#xB2A5;&#xD55C; lentiviruses (RCL)&#xC758; &#xC138;&#xB300;&#xB97C; &#xBC1C;&#xACAC; &#xD558; &#xACE0; &#xB3D9;&#xC6D0; &#xBCA1;&#xD130;. &#xC5F0;&#xAD6C;&#xD300;&#xC740; &#xB530;&#xB77C;&#xC11C; LV &#xD5A5;&#xC0C1; &#xB41C; &#xD6A8;&#xB2A5; &#xBC0F; biosafety &#xAE30;&#xB2A5; &#xC124;&#xACC4;&#xC5D0; &#xC0C1;&#xB2F9;&#xD55C; &#xB178;&#xB825;&#xC744; &#xBC14;&#xCE60; &#xACC4;&#xC18D;. &#xC2E4;&#xD5D8; &#xC5F0;&#xAD6C;&#xC5D0; &#xB300; &#xD55C; &#xD2B9;&#xC815; LV &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xC120;&#xD0DD;&#xC740; &#xC885;&#xC885; &#xC0DD;&#xC0B0;&#xC131; &#xBC0F; &#xBCC0;&#xD658; &#xD6A8;&#xC728; &#xC99D;&#xAC00; &#xD3EC;&#xD568; &#xD55C; &#xAE30;&#xB2A5;&#xC801; &#xACE0;&#xB824; &#xC0AC;&#xD56D;&#xC5D0; &#xC758;&#xD574; &#xAD6C;&#xB3D9; &#xB429;&#xB2C8;&#xB2E4;. &#xC548;&#xC804;&#xD55C; &#xBCA1;&#xD130; &#xB514;&#xC790;&#xC778;&#xB3C4; &#xC9C1;&#xC811; &#xC788;&#xB3C4;&#xB85D; &#xC5F0;&#xAD6C;&#xC6D0;&#xC740; &#xB354; &#xB0AE;&#xC740; &#xB9AC;&#xC2A4;&#xD06C;&#xC640; &#xC2E4;&#xD5D8; &#xC5F0;&#xAD6C;&#xB97C; &#xC218;&#xD589; &#xB3C4;&#xC6C0;&#xC774; &#xB418;. &#xD604;&#xC7AC;, &#xBCA1;&#xD130; &#xC99D;&#xAC00; &#xBCC0;&#xD658; &#xD6A8;&#xC728; &#xD5A5;&#xC0C1; &#xB41C; &#xC548;&#xC804; &#xAE30;&#xB2A5; (&#xC7AC;&#xC870;&#xD569; &#xBCA1;&#xD130; &#xB3D9;&#xC6D0;&#xC758; &#xC704;&#xD5D8; &#xAC10;&#xC18C;)&#xC744; &#xACB0;&#xD569; &#xD569;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xC704;&#xD5D8; &#xAD00;&#xB828; &#xB41C; &#xC140;&#xC758; &#xC774;&#xB7EC;&#xD55C; &#xBCA1;&#xD130;&#xC758; &#xC804;&#xBC18;&#xC801;&#xC778; &#xC704;&#xD5D8; &#xD3C9;&#xAC00;&#xC5D0; &#xD0D0;&#xC815; &#xC774;&#xB4DD; &#xD070; &#xC911;&#xC694;&#xC131;&#xC758; &#xC2E4;&#xC218;&#xB85C; &#xBCC0;&#xD658; &#xD558; &#xACE0; biosafety &#xC911;&#xC694; &#xD55C; &#xAD00;&#xC2EC;&#xC0AC;&#xAC00;. &#xC774; &#xAC80;&#xD1A0;&#xB294; LV biosafety &#xCD5C;&#xC2E0;&#xC758; &#xBE44;&#xAD50; &#xBC0F; &#xC2E0;&#xD765; LV &#xC0DD;&#xC0B0; &#xC2DC;&#xC2A4;&#xD15C; &#xD3EC;&#xD568; &#xB41C; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xBC1C;&#xC0DD;&#xD560; &#xC218; &#xC788;&#xB294; biosafety &#xBB38;&#xC81C;&#xB97C; &#xAC15;&#xC870; &#xD45C;&#xC2DC; &#xD558; &#xC5EC; &#xAC1C;&#xC120; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xB2E4;&#xB978; &#xC804;&#xB7B5;&#xC744; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. &#xBA87; &#xAE30;&#xC874; &#xAD6D;&#xB0B4;&#xC678; biosafety &#xAD8C;&#xACE0; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xB77C;&#xC2A4;&#xBCA0;&#xAC00;&#xC2A4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xC8FC;&#xC18C;&#xB97C; &#xC124;&#xBA85; &#xD558;&#xB294; &#xBC0F; &#xB77C;&#xC2A4;&#xBCA0;&#xAC00;&#xC2A4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xAC00;&#xC7A5; &#xC77C;&#xBC18;&#xC801;&#xC778; &#xC5F0;&#xAD6C; &#xD65C;&#xB3D9;&#xC5D0; &#xB300; &#xD55C; &#xAD8C;&#xC7A5; &#xC0AC;&#xD56D;&#xC744; &#xC81C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4;.

&#xC5F0;&#xAD6C; &#xC0AC;&#xC6A9;&#xC5D0; &#xB300; &#xD55C; &#xCD5C;&#xC2E0;&#xC758; lentiviral &#xBCA1;&#xD130;: &#xC704;&#xD5D8; &#xD3C9;&#xAC00; &#xBC0F; biosafety &#xAD8C;&#xC7A5; &#xC0AC;&#xD56D;.

Vectores lentivirales (LV) son veh&iacute;culos de transferencia g&eacute;nica competente, seg&uacute;n lo utilizado para aplicaciones de investigaci&oacute;n y gene terapia, debido a su integraci&oacute;n estable en no se dividen y dividiendo las c&eacute;lulas y expresi&oacute;n de transgenes a largo plazo. Junto con nuestra comprensi&oacute;n que LV ofrecen soluciones para la terapia g&eacute;nica, persiste la preocupaci&oacute;n han puesto al descubierto los riesgos debidos a insertional mutag&eacute;nesis, la generaci&oacute;n de lentivirus competente de replicaci&oacute;n (RCL) y vector de movilizaci&oacute;n. Por lo tanto, los investigadores siguen dedicando esfuerzos significativos en el dise&ntilde;o de LV con mayor eficacia y seguridad de la biotecnolog&iacute;a. La elecci&oacute;n de un sistema particular de LV para estudios experimentales a menudo est&aacute; impulsada por consideraciones funcionales, incluyendo mayor eficiencia productividad o transducci&oacute;n. El dise&ntilde;o de vectores m&aacute;s seguros tambi&eacute;n directamente ha beneficiado a los investigadores que les permita realizar estudios experimentales con menor riesgo. Actualmente, vectores combinan caracter&iacute;sticas de seguridad mejoradas (que disminuyen el riesgo de recombinaci&oacute;n y movilizaci&oacute;n del vector) con eficacia creciente de la transducci&oacute;n de se&ntilde;ales. Por lo tanto, los riesgos asociados con la transducci&oacute;n inadvertida de las c&eacute;lulas de la mayor importancia de investigador ganancia en evaluar el riesgo global de estos vectores y convertido en una preocupaci&oacute;n importante de bioseguridad. Esta revisi&oacute;n resume las diferentes estrategias para mejorar la bioseguridad LV comparando estado-of-the-art y emergentes de sistemas de producci&oacute;n de LV y resaltando temas de bioseguridad que pueden surgir durante su utilizaci&oacute;n confinada. Se discuten las pocas recomendaciones nacionales e internacionales de bioseguridad que tratan espec&iacute;ficamente el uso de LV en la investigaci&oacute;n y se proponen recomendaciones para actividades de investigaci&oacute;n m&aacute;s comunes usando LV.

Estado-of-the-art vectores lentivirales para uso de la investigaci&oacute;n: el riesgo de las recomendaciones de la evaluaci&oacute;n y bioseguridad.

CD4⁺ T cells are critical regulators of immune responses, but their functional role in human breast cancer is relatively unknown. The goal of this study was to produce an image of CD4⁺ T cells infiltrating breast tumors using limited ex vivo manipulation to better understand the in vivo differences associated with patient prognosis. We performed comprehensive molecular profiling of infiltrating CD4⁺ T cells isolated from untreated invasive primary tumors and found that the infiltrating T cell subpopulations included follicular helper T (Tfh) cells, which have not previously been found in solid tumors, as well as Th1, Th2, and Th17 effector memory cells and Tregs. T cell signaling pathway alterations included a mixture of activation and suppression characterized by restricted cytokine/chemokine production, which inversely paralleled lymphoid infiltration levels and could be reproduced in activated donor CD4⁺ T cells treated with primary tumor supernatant. A comparison of extensively versus minimally infiltrated tumors showed that CXCL13-producing CD4⁺ Tfh cells distinguish extensive immune infiltrates, principally located in tertiary lymphoid structure germinal centers. An 8-gene Tfh signature, signifying organized antitumor immunity, robustly predicted survival or preoperative response to chemotherapy. Our identification of CD4⁺ Tfh cells in breast cancer suggests that they are an important immune element whose presence in the tumor is a prognostic factor.

CD4⁺ follicular helper T cell infiltration predicts breast cancer survival.

By analyzing CD4(+) lymphocytes in human breast carcinomas, we have recently uncovered the presence of follicular helper T cells in lesions exhibiting an extensive immune infiltrate. The presence of these specialized CD4(+) T cells, which localize to the germinal centers of peritumoral tertiary lymphoid structures found in extensively infiltrated neoplastic lesions, predicts improved disease outcome among breast carcinoma patients.

Article	Year
Identification of three NFAT binding motifs in the 5'-upstream region of the human CD3gamma gene that differentially bind NFATc1, NFATc2, and NF-kappa B p50. The Journal of biological chemistry\| PubMed ID: 12374807	2002
Transcriptional regulation of the human CD3 gamma gene: the TATA-less CD3 gamma promoter functions via an initiator and contiguous Sp-binding elements. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 15879122	2005
6q- is an early and persistent chromosomal aberration in CD3-CD4+ T-cell clones associated with the lymphocytic variant of hypereosinophilic syndrome. Haematologica\| PubMed ID: 15951288	2005
Defective CD3gamma gene transcription is associated with NFATc2 overexpression in the lymphocytic variant of hypereosinophilic syndrome. Experimental hematology\| PubMed ID: 16219537	2005
Pertussis toxin activates adult and neonatal naive human CD4+ T lymphocytes. European journal of immunology\| PubMed ID: 16783847	2006
Bortezomib (PS-341, Velcade) increases the efficacy of trastuzumab (Herceptin) in HER-2-positive breast cancer cells in a synergistic manner. Molecular cancer therapeutics\| PubMed ID: 17148762	2006
Progressive loss of CD3 expression after HTLV-I infection results from chromatin remodeling affecting all the CD3 genes and persists despite early viral genes silencing. Virology journal\| PubMed ID: 17822534	2007
Molecular profiling of CD3-CD4+ T cells from patients with the lymphocytic variant of hypereosinophilic syndrome reveals targeting of growth control pathways. Blood\| PubMed ID: 19608752	2009
State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Current gene therapy\| PubMed ID: 20021330	2009
CD4⁺ follicular helper T cell infiltration predicts breast cancer survival. The Journal of clinical investigation\| PubMed ID: 23778140	2013
Tumor-infiltrating follicular helper T cells: The new kids on the block. Oncoimmunology\| PubMed ID: 24244900	2013

Karen Willard-Gallo

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