Bu çalışma, Hücresel Tedaviler (sözleşme NIH-NHLBI (HB-10-03) HHSN26820100000C) (CMR), Hücre tabanlı Terapi İhtisas Merkezleri Üretim Yardım tarafından desteklenen hibe NIH-NHLBI 1 U54 HL081007 (CMR), bir ASBMT Genç Araştırmacı Ödülü (UG ve OG), Klinik Araştırma Ödülü (UG) Lösemi ve Lenfoma Derneği Özel Üyesi ve Amy Strelzer Manasevit Scholar Ödülü (AML).

1. DC nucleofection <ol><li> Hücre Genix IL4 (1000U/ml) ile desteklenmiş Medya, GMCSF (800IU/ml) ve DC olgunlaşma sitokinler IL4 (1000U kullanarak 24 saat için daha olgunlaşmış kullanarak 5 gün boyunca plastik bağlılık kültürü ile zenginleştirilmiştir Hasat monosit türevli DC&#39;lerin, / ml), 3ml transfer pipet yardımıyla yumuşak tabanda GMCSF (800IU/ml), IL6100ng/ml, TNF-α 10 ng, IL1-β 10 ng ve PGE2 (1μg/ml) 1,. </li><li> Tripan mavi, en az 6 0.5x10 daha 3x 15ml tüp içine transferi ve daha 2x10 6 hücre / tüp kullanarak yaşayabilir DC&#39;ler Sayısı. </li><li> 10mins @ 200g DC&#39;ler Santrifüj. Üç kuyu, 37 ° C / 5% CO 2 inkübatör plaka tedavi 12 iyi doku kültürü 2ml/well - Bu süre içinde ön-sıcak Hücre Genix medya DC olgunlaşma sitokinler (DC olgunlaşma medya) ile desteklenebilir . </li><li> Hücreler bir kez iplik bitirdikten süpernatant aspirat ve 5μg DNA / tüp nihai bir konsantrasyon tüplerin her biri için ilgili DNA plazmid ekleyin. Bu durumda plazmid kodlama tüp IE1 pp65 # 1, tüp Hexon Penton # 2, ve tüp # 3 EBNA1-LMP2-BZLF1 ekleyin. </li><li> Amaxa nucloefection çözüm 100μl ile yeniden süspanse DC&#39;ler ve DNA, iyice karıştırın ve nucleofection küvetler transfer. </li><li> 4D nucleofector Yeri küvetler, program CB150 (Amaxa / Lonza) seçin ve start tuşuna basın. </li><li> Nucleofection küvet 2ml önceden ısıtılmış Hücre Genix DC olgunlaşma medya 500μl eklemek hemen sonra, prewarmed kalan 1.5 ml&#39;lik hazırlanan 12 plaka için aşağı yukarı 2-3 kez ve transfer nucleofected DC&#39;ler pipetleme ve hafifçe karıştırın DC olgunlaşma medya. 37 Transfer ° C /% 5 CO 2 inkübatör, bir başka 12-18hrs . </li></ol> 2. T hücre uyarımı <ol><li> Hasat ve 30Gy az nucleofected DC&#39;ler sayımı, ve ışın tedavisi. CTL orta 10ml ile (% 45 RPMI,% 45 Tıklanma EHAA,% 10 FBS, 2mm Glutamax) ve CTL medya ml&#39;de tekrar süspansiyon @ 3 x 10 5 DC&#39;ler ile bir kez yıkayın . </li><li> Havuz 7.5x10 5 (2.5ml) ve her içeren plazmid DC&#39;ler 15x10 5 (5ml), maksimum ve minimum toplanmış DC&#39;ler sonra kuvöz içinde yer alacak G-Rex cihaza aktarmak. </li><li> Cevapçı hücrelerinin hazırlanması için önceden dondurulmuş PBMC&#39;ler veya DC seçimi (yapışma veya CD14 seçimi) sonra kalan yapışmaz mononükleer hücreler ya kullanın. Hücrelerin çözülme, prewarmed kültür ortamına transfer CTL medya ile bir kez yıkayın. CTL Medya hücrelerinin yeniden süspanse hücre sayımı ve ml&#39;de 2x10 6 hücre konsantrasyonu getirin. Ve 300ng IL7 (10 ng - son derişim) - 30000U IL4 (son kons 1000U/ml) ile 15x10 6 hücre veya 7.5ml ve ek çekin. </li><li> PBMC 7.5ml (15x10 6 hücre), G-Rex ve medya ile toplam hacim 30ml bir CTL biyoreaktör kontör transferi . </li><li> Kültür G-Rex 6-7 gün süreyle 37 ° C /% 5 CO 2 nemlendirilmiş inkübatör. </li></ol> 3. T hücre genişletme <ol><li> Günde 6-7, medya aspirat 10ml, 10ml pipet ile medya kalan 20ml hücrelerin karışımı ve tripan mavisi kullanarak canlı hücreleri saymak. Varsa &lt;50x10 6 taze medya + sitokinler ile doldurmak. &gt; 50x10 6 hücre varsa, hücre süspansiyonu 10ml kaldırmak, yeni bir G-Rex transferi, ve sonra taze CTL medya + sitokinler ile hem de G-Rexs beslemek . </li><li> Kültür ek olarak 4-6 gün. Yeterli hücre ileride kullanmak üzere CTL ve cryopreserve fazla fenotipik ve fonksiyonel karakterizasyonu gerçekleştirmek genişletilmiştir. </li></ol> 4. Temsilcisi Sonuçlar: FDA onaylı multivirus spesifik CTL nesil sürecinin şematik Şekil 1&#39;de gösterildiği gibi, virüs-reaktif T hücreleri uyarmak için adenovectors ve EBV-LCL kongre multivirus CTL protokollerin aksine 2 DNA plazmid ile bulaşıcı bir virüs malzeme yerini almıştır 3 virüsleri her türetilen birden fazla antijene kodlamak. Biz üç multicistronic EBV plazmid kodlama Hexon ve Penton adenovirüs, IE1 ve pp65 CMV ve EBNA1, LMP2 ve BZLF1 trivirus CTL uyarmak için tasarlanmıştır. Bu antijenler, kendi ve T hücreleri İlan-hexon ve Penton 2,4-6 yöneltilmiş ve CMV-1E1 ve CMV pp65 in vivo 7 koruyucu gösteren diğer grupların klinik sonuçlar cesaret göre seçilmiştir. EBV, EBNA1 bir immünodominant CD4 + T hücre hedef antijen EBV ile ilişkili maligniteler ve EBV ile enfekte olan B hücreleri, normal ifade 8,9, LMP2 birden fazla HLA tipleri arasında immünojenik ve çoğu EBV maligniteler ifade 10,11 BZLF1 süre en bireyler hem CD4 + ve CD8 + T hücreleri uyarır ve işbirliği için büyük olasılıkla önemli olduğunu immünodominant, derhal erken litik döngüsü antijeni kodlayanvirüsü 12 kopyalayan hücreleri ntrol. Üretim yöntemleri daha da optimize etmek için CTL stimülasyon 13,14 minimalized, antibiyotik (FDA-uyumlu) plazmid oluşturulan Doğa Teknolojisi ile işbirliği yaptı. IFNγ ELISPOT ile ölçülen gibi optimize edilmiş DNA plazmid yanıt virüs spesifik T hücrelerinin sıklığı, daha fazla olduğunu, yüksek hücre canlılığı (veriler gösterilmemiştir) 3. Şekil 2 korurken, bu stratejiyi kullanarak sürekli olarak&gt;% 35 nucleofection verimi sağlamak aynı antijenleri (n = 8 Adenovirüs, n = 4, CMV ve n = 2 EBV) ifade geleneksel pShuttle tabanlı ifade plazmid yanıt daha. DC optimal oranı: R oranı (n = 2 vericileri): hücre, Şekil 3&#39;te 1:50 &#39;lik bir oran 1:20 G göre sub-optimal aktivasyon görüldüğü gibi güçlü bir T hücre uyarılması için önemliydi . Üretim geniş antijen özgüllüğü yeterli CTL numaraları üç virüslere karşı klinik etkinliği için bir ön koşuldur. Bu, kültürleri artar IL4 ve IL7 ek olarak repertuar ve özgüllüğü ise Şekil 4B gösterilen, geleneksel 24-kuyucuğu (Şekil 4A) 15 ile karşılaştırıldığında üstün T hücre genişlemesini destekliyor G-Rex, CTL kültürü ile elde CMV pp65-türevli HLA-A2 karşı reaktif T hücrelerinin sıklığı resticted NLV peptid IL4 ve / veya IL7 16,17 varlığı ya da yokluğu oluşturulan kültürlerde değerlendirildi. Dondurulması / infüzyon için nihai ürün üzerindeki akım sitometri analizi gerçekleştirmek genişletilmiş hücreleri, hücre içi sitokin boyama / IFNγ ELISPOT, ve Cr 51 serbest deneyleri fenotip ve fonksiyonel kapasitesini değerlendirmek için. Tipik olarak oluşturulan hücreler CD4 + ve hem de T hücre bölümlerinde algılanabilir antijen-özgüllük ile CD8 + T hücreleri karışık bir nüfusa sahip poliklonal. CTL in vivo (Şekil 5) graft-versus-host hastalığı (GVHH) neden olmaması gerektiğini belirterek, virüs kısmen HLA uyumlu hedefler negatif viral antijen-ifade hedef hücreleri öldürmek ancak bunlarla. <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="Şekil 1"/> Şekil 1 rCTL ​​nesil protokol. Birincisi, DC&#39;ler viral antijen-kodlama plazmid nucleofected ve ardından otolog PBMC bir R ile karışık: S 10 veya 20:01. Hücreler, sonra hasat edilmiş sayılır, işlev, kimlik ve sterilite için test ve daha sonra klinik kullanım için Kriyoprezerve IL4 ve IL7 varlığı için 10-14 gün, G-Rex genişletilmiştir. <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="Şekil 2"/> Şekil 2. Optimize edilmiş DNA plazmid, in vitro üstün T hücre aktivasyonuna neden olur . DC&#39;ler optimize edilmiş, FDA uyumlu plazmid kodlama Hexon ve Penton (İlan), IE1 ve pp65 (CMV) ve EBNA1, LMP2 ve BZLF1 (EBV) veya aynı antijenleri kodlayan geleneksel pShuttle plazmid nucleofected. Bu T hücreleri uyarmak için kullanılır ve özgüllük IFNγ ELISPOT 10 gün stimülasyon ile analiz edildi. <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="Şekil 3"/> Şekil 3 Optimal DC: CTL aktivasyonu için T hücre oranları. Üç optimize plazmid 2 vericilerden DC&#39;ler nucleofected ve sonra, 01:20 ya da 01:50 DC otolog PBMC teşvik etmek için kullanılır: PBMC oranı. IFNγ ELISPOT tarafından 10 günde yeniden T hücrelerinin sıklığı değerlendirildi. <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="Şekil 4"/> Şekil 4 artırılması sitokinler kullanarak G-Rex T hücre genişletme. Panel A, G-Rex cihaz yanı sıra mikroskopi tarafından değerlendirilir gaz geçirgen zar, CTL görünüm gösterir. Plakalar vs G-Rex tedavi kongre doku kültürü elde hücre çıkış arasında bir karşılaştırma da gösterilmiştir. B Paneli CMV pentamer olumlu CTL sitokin, tek başına IL4, IL7 yalnız ve IL4 + IL7 varlığı genişledi kültürleri elde sıklığını gösterir. <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="Şekil 5"/> Şekil 5. Genişletilmiş CTL fenotipi ve fonksiyonu. Ve CD8 + - Panel A CD4 + (yardımcı 45%) karışımı ile poliklonal genişletilmiş multivirus CTL, fenotipi bir temsili örnek gösterir (% 42 - sitotoksik) çoğunluğu (% 95) ifade olan T hücreleri, bellek işaretleyici CD45RO + / CD62L +. Paneli Yatak bu hücrelerin tüm uyarıcı antijenleri için spesifik olan ve IFNΓ, üretim ve antijen uyarımı sonrası TNF tespit hücre içi sitokin boyama tarafından değerlendirilir olarak polifonksiyonel vardır ki gösterir. Panel C genişletilmiş CTL ölçülen işlevsel olduğunu gösteren Cr 51 yöntemi ile. Otolog LCL, tek başına ya da CMV pp65 ifade eden bir boş vektör ya da bir adenoviral vektör ile transduced tar olarak kullanıldıolur. Alloreactivity allojenik PHA patlamalar kullanarak bir hedef olarak değerlendirildi.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N. Engl. J. Med. 333, 1038-1044 (1995).</li></ol>

Juan F. Vera Wilson Wolf Üretim için bir danışman. Diğer yazarlar ilgili açıklamalar var.

Viral enfeksiyonlar, hastalığı veya tedavisi immün sistemi baskılanmış hastalarda önemli bir morbidite ve mortalite oluşturmaktadır. HSCT sonra, örneğin, EBV ve CMV yanı sıra Respiratuvar Sinsityal Virüs (RSV) gibi solunum yolu virüsleri gibi kalıcı herpesvirüslerinin kaynaklanan enfeksiyonlar, iyi bilinen, İlan, BK virüs neden olduğu enfeksiyonların önemi ve insan herpes virüs (HHV) -6 daha takdir edilmiştir. Farmakolojik ajanların bazı enfeksiyonları için standart tedavi olmasına rağmen, önemli toksisiteleri, dayanıklı türevleri üretmek, ve sık sık etkisiz kalmaktadır. Buna karşın, kök hücre donörlerden elde edilen virüs spesifik T hücrelerinin hemopoietik kök hücre nakli, viral enfeksiyon veya hastalık (HSCT) ayarı 2,5,6,18-21 önlenmesi ve tedavisi için güvenli ve etkili olduğu kanıtlanmıştır. Ancak, T hücre immünoterapi geniş bir uygulama sonuçta CTL üretimi için gerekli olan maliyet, karmaşıklık ve zaman ile sınırlıdır. Multivirus CTL, mevcut elyazması açıklanan oluşturmak için yeni ve hızlı bir yaklaşım önemli ölçüde bağışıklığı baskılanmış konak için standart bir tedavi haline strateji sağlayarak, sitotoksik T hücre tedavisi viral hastalıklar için fizibilite artırmanız gerekir. APC&#39;ler plazmid nucleofected DC&#39;ler kullanımı farklı DC popülasyonları her plazmid 3 için kullanılmaktadır beri tek bir hücre içinde ifade birden çok viral antijenler viral vektörler ya da gerçekten rekabet olmadan MHC sınıf I ve II antijen sunumu sağlar. IL-4 / 7 artar, buna 16,17 antijen-spesifik T hücrelerinin yanıt sıklığı ve repertuar artmasına yardımcı olur T hücre sağkalım ve proliferasyon, kullanın . Son olarak, G-Rex kültürü önemli ölçüde kültür sırasında T hücre apoptozis azaltır. Gaz değişimi (O 2 ve CO 2 çıkışı), hücreler üzerinde daha derin bir orta izin verirken hipoksi önlenmesi, daha fazla besin ve atık ürünlerin seyreltilmesi, balonun tabanındaki bir gaz geçirgen silikon zarından oluşur . Bu platform, aynı zamanda ek virüslere karşı koruyucu antijenler olarak tanımlanır uzatılabilir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005 </td></tr><tr><td> IL4 </td><td> R + D Systems </td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td> Peprotech </td><td> 200-15 </td></tr><tr><td> Hyclone RPMI 1640 </td><td> Thermo Scientific </td><td> SH30096.01 </td></tr><tr><td> İnsan AB serum </td><td> Valley Biyomedikal Inc. </td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa / Lonza </td><td> AAF-1001B &amp; AAF-1001X </td></tr><tr><td> Nucleofection Kiti </td><td> Amaxa / Lonza </td><td> V4XP-3012 </td></tr><tr><td> Plazmidler </td><td> NTC </td><td> n / a </td></tr><tr><td> GM-CSF </td><td> Ar-Ge </td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td> Ar-Ge </td><td> 201-LB-025 </td></tr><tr><td> IL-6 </td><td> Ar-Ge </td><td> 206-IL-CF </td></tr><tr><td> TNF </td><td> Ar-Ge </td><td> 210-TA-010 </td></tr><tr><td> PGE2 </td><td> Sigma </td><td> P6532-1MG </td></tr><tr><td> G-Rex </td><td> Wilson Kurt Üretim </td><td> AY11-00.027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

Viral enfeksiyonlar, allojeneik hematopoietik kök hücre transplantasyonu (HKHT) alıcıları morbidite ve mortalite neden olur. Biz ve diğerleri başarılı bir şekilde oluşturulur ve Ebstein Barr virüsü (EBV), sitomegalovirüs (CMV) ve Adenovirüs (İlan) monositler ve T-hücrelerinin özel infüze EBV-dönüştürülmüş lenfoblastoid hücre (EBV-LCL) adenovirüs vektör ile gen-modifiye antijen sunan hücreler (APC). / Kg gibi az 2x10 5 trivirus spesifik sitotoksik T lenfositler (CTL) infüzyon birkaç günlükler sonra çoğaldı ve mevcut diğer tedavilere dirençli, hatta ciddi bir viral hastalıktır önlemek ve tedavi etmek için ortaya çıktı. Hazırlanması için geniş bir uygulama bu teşvik edici bir yaklaşım, yüksek üretim maliyetleri, üretim karmaşıklığını ve uzun bir süre (toplam 10-14 hafta CTL üretimi için EBV LCL nesil, ve 4-8 hafta 4-6 hafta) ile sınırlıdır. Bu sınırlamaları aşmak için yeni bir GMP uyumlu CTL üretim protokolü geliştirdik. Birincisi, biz, antijen sunan plazmid DNA kodlaması LMP2, EBNA1 ve BZLF1 (EBV), Hexon ve Penton (İlan), pp65 ve IE1 (CMV) ile nucleofected dendritik hücreler (DC) T hücreleri uyarmak için adenovectors yerine hücreleri. Bu APC tüm uyarıcı antijenler için spesifik T hücrelerinin yeniden etkinleştirin. İkincisi, kültür varlığında IL-4 T-hücrelerinin aktive (1.000 U / ml) ve IL-7 (10 ng) artırır ve bizim çizgiler spesifik T hücrelerinin repertuar ve frekans sürdürmektedir. Üçüncü olarak, biz, böylece EBV-LCLs şartının kaldırılması ve teknisyen müdahale azaltarak, tek bir stimülasyon sonra büyük hücre sayıları genişleme ve hayatta kalma teşvik yeni bir gaz geçirgen kültür cihazı (G-Rex) kullanılır. Bu değişiklikler uygulayarak şimdi 10 6 hücre&gt;% 90 azalır başı maliyetle, EBV, CMV, ve İlan hedefleyen multispecific CTL üretmek ve ek için uzatılabilir bir yaklaşım kullanarak sadece 10 gün yerine 10 hafta Koruyucu viral antijenler. Değeri yüksek risk allojenik HKHT alıcıları için profilaktik ve tedavi uygulamaları için FDA onaylı bir yaklaşım olmalıdır.

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Üretimi için hızlı, basit ve maliyet-etkin bir protokol, CMV, İlan EBV enfeksiyonları yakalanma riski altında allojeneik hematopoietik kök hücre nakli (HSCT) alıcıları infüzyon için donör kaynaklı multivirus spesifik CTL (rCTL). Bu üretim sürecine GMP uyumlu ve T-hücre immünoterapi uzmanlaşmış merkezlerde ötesinde daha geniş bir şekilde uygulanmasını sağlamak olmalıdır.

Multivirus-spesifik T hücrelerinin üretilmesi Allojenik Hematopoetik Kök Hücre Nakli sonra Viral Enfeksiyonlar önleme / tedavi etmek için

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

generation-multivirus-specific-t-cells-to-preventtreat-viral

Research

JoVE Journal

Immunology and Infection

17.1K Görüntüleme.  Baylor College of Medicine. Üretimi için hızlı, basit ve maliyet-etkin bir protokol, CMV, İlan EBV enfeksiyonları yakalanma riski altında allojeneik hematopoietik kök hücre nakli (HSCT) alıcıları infüzyon için donör kaynaklı multivirus spesifik CTL (rCTL). Bu üretim sürecine GMP uyumlu ve T-hücre immünoterapi uzmanlaşmış merkezlerde ötesinde daha geniş bir şekilde uygulanmasını sağlamak olmalıdır.

Video: Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

İmmünoloji ve Enfeksiyon

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

İlköğretim İnsan naif ve Bellek T Hücreleri gelen İsteyerek Düzenleyici T Hücreleri Üretimi

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Klinik Uygulama<em&gt; Sleeping Beauty</emGenetiği Periferik ve Göbek Kordon Kanı alınan T hücreleri Değiştir Sunan Hücreler&gt; ve Yapay Antijen

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

Saf Hematopoetik kök / progenitör hücrelerin DNA mutasyonları belirlenmesi

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Farelerde allojenik CD4 + T hücre Cevapları Eğitim için Kuyruk Cilt Nakli

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Sitokin yakalama Sistemi kullanılarak Klinik Uygulamalar için Sitomegalovirüs özgü otomatik Hücre Zenginleştirme T hücreleri

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Üretim ve Çok fenotipik Yüksek içerik Eleme<em&gt; Coxiella burnetii</em&gt; Transposon Mutantlar

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Otoimmünite karşı Kök Hücre türetilmiş antijen-özgü düzenleyici T hücrelerinin gelişimini

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Görüntüleme Akım Sitometri Kullanımı T Hücresi alloreaktivitesini Ölçümü

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Virüslü Ifnar1- / - farelerin Immunoprivileged organlarında Zika virüs özgü T hücreli yanıt-e doğru değerlendirilmesi

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...