We thank Miltenyi Biotec, Germany for providing reagents and CliniMACS Prodigy equipment for evaluation studies. We thank George T. McNamara (Pediatric department, MD Anderson Cancer Center) for proof reading the manuscript. Grant support: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303; CA141303); R33 (CA116127); P01 (CA148600); Burroughs Wellcome Fund; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Estate of Noelan L. Bibler; Gillson Longenbaugh Foundation; Harry T. Mangurian, Jr., Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC&#8217;s Sister Institution Network Fund; Miller Foundation; Mr. Herb Simons; Mr. and Mrs. Joe H. Scales; Mr. Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; William Lawrence and Blanche Hughes Children's Foundation.

Steril Koşullarında Malzemelerin hazırlanması 1. (Malzemeleri ve Ekipmanları Tablo bakınız) <ol><li> % 0.5 bir son konsantrasyona kadar insan serum albümini (HSA) ile desteklenmiş PBS / EDTA tampon maddesi 3 L hazırlanması (a / h). </li><li> Klinik sınıf% 0.9 sodyum klorid (NaCl) çözeltisi 1 L&#39;lik bir çanta GMP sınıfı hücre kültür ortamının 2 L hazırlayın. </li><li> Steril su, 8 ml CMV pp65 bir vial sulandırılması CMV-spesifik peptid antijeni kokteyli 60 nmol hazırlayın. </li><li> Bir Luer / Spike interconnector kullanılarak 50 ml hacim dondurucu torbaya CMV pp65 peptid kokteyl aktarın ve boru setinin içine kokteyl sonraki dağılımını önlemek için forseps kilitleme kelepçe. Steril koşullar altında Açık hücreli zenginleştirme boru seti (TS 500). </li><li> Steril boru kaynakçı kullanarak, şu anda peptit kokteyl çantası kelepçesini açmayın boru setinin vananın 2 TS 500 için tüp bağlantısı içine peptid kokteyl donma çanta bağlayın. </li><li> Kaldır 1 x 10 9 TBaşlangıç ​​hücre üründen NC ve 50 ml &#39;lik bir toplam hacme% 2.5 HSA ihtiva eden PBS / EDTA tampon maddesi içinde askıya. 150 ml&#39;lik bir aktarma torbaya hücresel ürünü enjekte edilir. </li></ol> 2. Hazırlık ve Kullanım otomasyon Hücre Zenginleştirme Sistemi (Malzemeleri ve Ekipmanları Tablo bakınız) <ol><li> Hücre zenginleştirme sistemi (Şekil 1B) açın ve programı &quot;CCS_IFN-γ Zenginleştirme&quot; seçeneğini seçin. Prosedür yoluyla operatöre rehberlik talimatlar ve resimlerle ekranları gösteren bir kullanıcı arayüzü gözlemleyin. </li><li> Parametresi &quot;Operatör&quot; ve &quot;Boru Seti P / N Hayır&quot; girin. Sonraki interaktif monitör ekranında görüntülenen talimatlara göre Boru Seti 500 otomatik hücre zenginleştirme cihazı takın. </li><li> Cihaza orta ve tamponlar bağlamak için ekranda görüntülenen yönergeleri adım adım izleyin. Kayıt katalog numarası ve connectin önce reaktiflerin lot numarasıCihaza örn. </li><li> Boru setinin son kontrolünden sonra, peptid kokteyl çanta kelepçeyi açın. Orta çantayı açın ve boru setinin otomatik hazırlanmasını başlatmak. </li><li> Priming adımı tamamlandıktan sonra, steril boru kaynakçı yardımıyla rezervuar torba (200 mi) içinde NaCI tampon içine HSA (% 2.5) tamamlar. Steril boru kaynak makinası kullanarak &quot;Uygulama bag&quot; içine başlayan hücresel ürünü aktarın. </li><li> Adaptörleri aracılığı ile ilgili boru içine CCS (IFNy) reaktifler bağlayın. Zenginleştirme işlemi öncesinde selüler malzemenin bir kısmını toplamak için tercih edilen bir zaman girin. İnceleme ve tüm verilerin doğruluğunu / parametreler girilir. Işlemini başlatın. </li><li> Otomatik hücre zenginleştirme işlemi başlamadan önce, kalite kontrol yastığı (QCB orijinal fraksiyon (ori) PBS / EDTA tampon maddesi ile seyreltildi, 100 mi odacık içeriği üzerinden yaklaşık 1.3 ml içeren) çıkarın. , QCB Seal tartmak ve 4 ° C&#39;de saklayın. </li><li> Enrichme başlatnt süreci. İşlemin sonunda, hedef hücreler, rezervuar torba gelen seçme tampon çözeltisi yaklaşık bir hacmi ile elüt edilecektir. </li><li> Olmayan Hedef Hücre Bag (NTCB, negatif fraksiyon = negatif) ve Hedef Hücre Çanta (TCB, pozitif kesir = pos) Mühür ve her torbayı tartın. Ağırlıklar daha sonra hücre sayıları hesaplanması için kullanılacaktır. </li><li> Hemen zenginleştirme işleminden sonra akış sitometresi analizi için fraksiyondan başına iki alikotları toplamak ve 4 ° C&#39;de numunelerin kalan saklayın. Hücre sayısı tayin zenginleştirme performans analizi (Tablo 1) Diğer numune kısım için bir numune kısım kullanın. </li><li> Hücresi zenginleştirme aletten boru setinin çıkarın. Ileride kullanmak üzere bir USB sürücüsüne günlük dosyasını aktarın. Not: Tüm reaktifler, steril koşullar altında hazırlanmalıdır. Biyogüvenlik tip II kaput kullanılması tavsiye edilir. Sağlıklı izole kararlı durum aferezi hücresel ürün (non-mobilize) kullanarakCMV seropozitif verici CMV antijen spesifik T hücrelerini zenginleştirmek. Sadece FDA HSA kullanılmalıdır lisanslı. Hücre hazırlama tamponu indirgenmiş saflık ve bir hedef hücre, indirgenmiş verimle sonuçlanır düşük veya daha yüksek çevre sıcaklıklarında olarak 25 ° C&#39;ye kadar 19 ° C &#39;de tutulması gerekmektedir. </li></ol> 3. Hücre Sayısı Tayini <ol><li> Tablo 1 &#39;de gösterildiği gibi. Hücre sayımları için QCB, NTCB ve TCB hacimde al, 4 ° C&#39;de 10 dakika süreyle karanlıkta her kısım (titre 01:11) CD45-VoBlue ekleyin ve inkübe edilir. </li><li> Orijinal fraksiyonu ve negatif fraksiyon pozitif fraksiyona 450 ul taze hazırlanmış kırmızı kan hücre parçalama çözeltisi 1,5 ml taze hazırlanmış kırmızı kan hücresi lizis çözeltisi (1x) ekleyin ve oda sıcaklığında 15 dakika seyahati kesirler inkübe edin. </li><li> (100 ug / ml, 100 seyreltme 1) hemen önce analiz için 1 ug / ml nihai konsantrasyona kadar propidyum iyodür ilave edin. Hücre sayımı ve vi belirlemek için otomatik hücre sayacı kullanınyeteneği. Flow sitometri analizi için hücre karşı cihaz tavsiye yazılımını kullanın. Orijinal negatif ve pozitif fraksiyonları lökositlerin mutlak sayılarını belirlemek. Not: Hücre sayısı analizi için alınan örnekler ml&#39;si başına canlı hücre sayısı lökositlerin hücre analizörü tarafından önerilen yazılım kullanılarak belirlenir. </li><li> Şekil 2 (bölge 5, uygun bir lökositler) &#39;de gösterildiği gibi bölge ayarlayın. Hücre sayısını belirlemek için aşağıdaki yolluk strateji kullanın. Orijinal fraksiyonunda bir canlı lökosit Şekil 2 &#39;de gösterilmiştir. </li><li> Aşağıda belirtilen bölgeler (Şekil 2, 1-6) hiyerarşik olarak şunlardır: 1: Zaman kapısı → 2: Tek hücre → 3: CD45 + hücreler → 4: lökositler (enkaz hariç) 5 →: geçerli lökositlerin → 6: geçerli lenfositler </li><li> Negatif ve pozitif kesirler hücre sayılarını belirlemek için aynı adımları tekrarlayın. Bütün fraksiyonun hücre sayısını hesaplayınörnek ve fraksiyon (Tablo 2) toplam hacmine seyreltici faktörü dikkate alınarak. </li></ol> Ayırma Performansının 4. Ara Sınavı <ol><li> Önceden soğutulmuş PBS / EDTA tampon /% 0.5 AB serumu ile QCB, NTCB ve TCB fraksiyon hücrelerin alikotları yıkayın. 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de santrifüj ve hücreler süpernatan aspire. </li><li> Içeren 100 ul antikor florokrom boyama karışımı içinde süspanse edin hücreleri: CD3-FITC CD4-APC, CD8-APC-Vio770, CD14-PerCP, CD20-PerCP, CD45-VioBlue ve anti-IFNy-PE (titresi 01:11) ve 4 ° C&#39;de 10 dakika süreyle karanlıkta inkübe edilir. </li><li> 1 ml taze hazırlanmış kırmızı kan hücresi lizis çözeltisi (1x) ekleyin ve oda sıcaklığında 15 dakika inkübe edilir. 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de santrifüj ve süpernatan aspire. PBS / EDTA tampon /% 0.5 AB serumu yeterli bir hacimde tekrar süspansiyon hücreleri. </li><li> 100 diluti: 1 ug / ml hemen önce analiz (1 bir son konsantrasyona propidiyum iyot ekleme) 100 ug / ml üzerine. Numunenin saflığı değerlendirmek için flow sitometri analizi gerçekleştirir. </li><li> CCS zenginleştirme işleminden sonra pozitif fraksiyonun gösterilmiştir CD3 + T hücreleri belirlemek için uygun bir lökosit Yolluk stratejisinde CD3 + T hücreleri hesaplamak için aşağıdaki yolluk strateji kullanın. Aşağıda belirtilen bölgeler (Şekil 3A ve 3B, 1-6) hiyerarşik olarak bağlıdır: 1: Zaman kapısı → 2: Tek hücre → 3: CD45 + hücreler → 4: Hücreler (enkaz hariç) 5 →: geçerli lökositler → 6: geçerli CD3 + hücre popülasyonu </li><li> CD4 +, CD8 +, CD4 + IFN-y + ve CD8 + IFN-y + CCS zenginleştirme işleminden sonra, T hücreleri (Tablo 2) frekanslarını belirlemek. </li><li> F aşağıda CD4 +, CD8 +, CD4 + IFN-y + ve CD8 + IFN-y + T hücrelerinin frekansları belirlemek için yolluk strateji kullanarakveya CCS sürecinden sonra orijinal ve zenginleştirilmiş (esir) pozitif kesir. Aşağıdaki gibi gösterilen bölgeler hiyerarşik bağlantılı ve adlandırılır: 1: Zaman kapısı → 2: Tek hücre → 3: CD45 + hücreler → 4: Hücreler (enkaz hariç) 5 →: geçerli Lökosit → 6: geçerli CD3 + hücreleri → 7: CD4 + hücreleri → 7a: CD4 + IFN-γ + Hücreler (kutu) → 8: CD8 + hücreleri → 8a: CD8 + IFN-γ + hücreleri (kutu) </li></ol> NOT: hiyerarşi birinci bağlantı 6 belirtilen bölgeler, (1-6), burada Şekil 3 ile aynıdır ve 2 bölgeler geçen Şekil 4 (6-8a) &#39;de gösterilmiştir.

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MD Anderson Kanser Merkezi ve Cooper Hem ZIOPHARM Onkoloji, Inc., ve Intrexon Corporation mali ilgi var. 7 Mayıs 2015 tarihinde, Dr. Cooper ZIOPHARM Onkoloji Baş Müdür olarak atandı. Doktor Cooper şimdi, MD Anderson bir Misafir Araştırmacı olduğunu. Doktor Cooper kurulan ve o yapay nükleazlarla ile Sangamo BioSciences ile patente sahiptir InCellerate, Inc. sahibi. O Targazyme, Inc. (eski Amerikan Kök hücreler, Inc.), GE Healthcare, Ferring İlaç, Kader Therapeutics, Janssen Pharmaceuticals ve Bristol-Myers Squibb danışır. O Cellectis Bilimsel Danışma Kurulu üzerindedir. O, Miltenyi Biotec honoraria alır.

Evlatlık T-hücre tedavisi B-hücresi maligniteler 4 tedavisinde uygulanabilir bir seçenek olarak ortaya çıkmıştır. Bu tedavi edici potansiyeli replikatif yaşlanmayla 2 barındırmayan hedef antijene özel T hücreleri istenen sayıda beslerken bağlıdır. Bu halen geçerli gıda işleme uygulamalarıyla uyumlu genişletilmiş T hücrelerinden antijen belirli T hücrelerinin saf bir nüfus ayrıştırılması ile elde edilebilir. İki ayırma işlemleri yaygın olarak, yani, floresans ile ...

Hematopoietik kök hücre nakli (HSCT) 1, graft-versus-tümör etkisini arttırmak ve fırsatçı enfeksiyonlara 2 bağışıklığı sağlamak için adoptif T-hücre terapisi ile kombine edilebilir. Infüzyon için antijene spesifik donör kaynaklı T hücrelerinin üretilmesi tarihsel kalifiye personel ve GMP uyumlu olan uzman tesislerin kullanılmasını gerektirmiştir. Bu T hücrelerinin dağıtım fırsatçı enfeksiyon 3 çözünürlüğü hem de altta uzanan malignite 4 tedavi ile sonuçlanmıştır. Yakın geçmişte, araştırmacılar göstermiştir ki bin az sayıda virüs spesifik T hücrelerinin adoptif transfer (~ 1 x 10 4-2,5 x 10 5 hücre / kg alıcı vücut ağırlığı) başarılı bir şekilde HKHN 5-9 sonra fırsatçı CMV enfeksiyonları tedavi olabilir. Ilişkili vasıflı imalat şartlarına GMP tesislerinin sınırlı sayıda ve hücre üretimi ile ilişkili yüksek maliyetli, ancak, restr vardırT-hücre tedavileri 10 vaat için icted hasta erişim. Antijen-spesifik T hücrelerinin izole edilmesi için bir yaklaşım, CD45 ve IFN-y tanımak için bir çift spesifik bir reaktif kullanılarak CCS dayanmaktadır. Gösterildiği gibi, bu yöntem, bir otomatik hücre ilave CCS cihazı (Şekil 1B) kullanılarak klinik düzeyde CMV-spesifik T hücreleri üretmek için kullanılabilir. CMV-spesifik T hücreleri, CMV-seropozitif donörlerden lökaferez, toplam nükleer hücreler (TNC) CMV pp65 antijeninden örtüşen peptidlerin inkübe edilerek oluşturulur. İnsan lökosit antijen (HLA) bağlamında gösterildi Bu peptidler, IFN-y salgılaması için TNC olan CMV pp65-spesifik T hücrelerini aktive etmektedir. Bu T hücreleri daha sonra &quot;esir&quot; olmak ve manyetik ayrılmış olabilir. Birinci nesil cep zenginleştirme cihazının çalışması (Şekil 1A) birden s üstlenmek GMP koşulları altında hücre kültüründe yetişmiş personel gerekli ve personelin koordinasyonuBir &quot;esir&quot; ürünü üretmek için gerekli Teps. Prosedür genellikle sürekli çalışma 12 saat için 10 gerekli ve bu nedenle personelin büyük olasılıkla GMP tesisi iki vardiya üzerinden çalışmak gerekiyor. Bu kısıtlamalar, artık (Şekil 1B de gösterilen), bir ikinci kuşak cihazının uygulanması ile ortadan kaldırılır. Bu cihaz, ilk nesil cihaza benzer manyetik zenginleştirme, taahhüt, ancak bir unbreached bir yaklaşımla CCS&#39;nin diğer yönlerini otomatik hale getirir. Bu önemli ölçüde adımların en personeli tarafından sahipsiz yapılabilir olarak GMP ekibi üzerindeki yükü azaltır. Cihaz, bir kapalı bir sistem olarak işlev Dahası, antijen-spesifik T hücrelerinin yakalanır ve cihazı başlamadan önce lökaferez izolasyonu ve materyallerin hazırlanmasında yer alan adımları dışında masa üstü işlenebilir. Tam enstrümantasyon ve bu ikinci nesil cep zenginleştirme cihazın işlevselliği Detayları pub edilmiştir11 yayımlanır. Burada, biz otomatik hücre zenginleştirme CCS sistemini kullanarak bir kararlı durum aferez üründen CMV pp65-spesifik T hücrelerini zenginleştirmek için gereken adımları açıklar. Bir kez izole edildiğinde, bu, CMV-spesifik T hücreleri, hemen bir hastaya infüzyon yoluyla olabilir.

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			<td>CliniMACS PBS/EDTA Buffer 3 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>700-29</td>
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			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-182</td>
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			<td>5 L waste bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>110-004-067</td>
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			<td>Miltenyi Biotec GmbH</td>
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			<td>Miltenyi Biotec GmbH</td>
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			<td>Miltenyi Biotec GmbH</td>
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			<td>Water for injections</td>
			<td>Hospira, inc, Lake Forest, IL</td>
			<td>NDC-0409-4887-10</td>
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			<td>Millipore</td>
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			<td>TexMACS GMP Medium 2 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-306</td>
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			<td>Transfer Bag, 150 mL (for cellular starting material)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-301</td>
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			<td>Miltenyi Biotec GmbH</td>
			<td>200-074-400</td>
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			<td>BD, Laagstraat, Temse, Belgium</td>
			<td>309653</td>
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			<td>CMV sero positive apheresis product</td>
			<td>Key Biologics, LLC, Memphis</td>
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			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-065</td>
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			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-072</td>
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			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-077</td>
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			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-136</td>
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			<td>aIFN-&#947;-PE, human</td>
			<td>Miltenyi Biotec GmbH</td>
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			<td>Miltenyi Biotec GmbH</td>
			<td>130-091-374</td>
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			<td>Miltenyi Biotec GmbH</td>
			<td>130-093-233</td>
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			<td>CliniMACS Prodigy Device&#160;</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
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			<td rowspan="2">Miltenyi Biotec GmbH</td>
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			<td>22331</td>
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			<td>Nexelom Bioscience, Lawrence, MA</td>
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automated cell enrichment of cytomegalovirus-specific t cells for clinical applications using the cytokine-capture system

Patogene-özgü T hücrelerinin adoptif transfer önlenmesi ve allogeneik kök hücre transplantasyonundan sonra ortaya çıkan gibi enfeksiyon (CMV), sitomegalovirüs gibi fırsatçı enfeksiyonları tedavi etmek için de kullanılabilir. Üçüncü şahıs bağışçı olmak üzere allojenik vericilerden, viral-spesifik T hücreleri, seçici istenen T hücrelerini yaymak için antijen-odaklı uyarım tekrarlanan mermi kullanan mevcut iyi üretim uygulamaları (cGMP) uygun olarak ex vivo olarak yayılan olabilir. Antijen-spesifik T hücrelerinin tanımlanması ve izolasyonu da gama-interferon (IFN-γ) salgılamaya aktive edilmiş T hücrelerinin sitokin yakalama sistemine dayalı yapılabilir. Üretim süreci zaman alıcı ve yetenekli bir operatör gerektirir Ancak, bağışıklık geri yardımcı olmak için sitokin yakalama sistemi (CCS) yaygın insan uygulaması sınırlı kalmıştır. Şimdi bu CliniMACS Prodigy gibi ikinci nesil hücresi zenginleştirme cihazının geliştirilmesiotomatik bir, daha az emek yoğun bir sistem kullanılarak viral özgü T hücrelerinin üretilmesi için araştırmacılar sağlar. Bu cihaz, manyetik klinik sınıf ürünler üretmek için bir manyetik etkinleştirilmiş hücre tasnif teknolojisi kullanılarak etiketlenmemiş hücrelerden hücreler etiketli ayırır, kapalı bir sistem olarak tasarlanmıştır ve masa üstü erişilebilen ve çalıştırılabilir. Bu yeni otomatik hücresi zenginleştirme Cihazın çalışması, bir CMV seropozitif vericiden elde edilen bir kalıcı-hal aferez ürününden elde edilen CMV pp65-spesifik T hücreleri üretmek için göstermektedir. Bu izole edilmiş T hücreleri, daha sonra doğrudan, kurumsal ve federal düzenleme denetimi altında bir hastaya infüze edilebilir. Kırmızı kan hücrelerinin ortadan kaldırılması dahil olmak üzere tüm biyo-işlem aşamaları, T hücreleri, antijen-spesifik T hücrelerin, saflaştırma ve yıkama ayrılmasının uyarılması tam olarak otomatikleştirilmiştir. Bu gibi cihazlar, insan uygulaması için T hücreleri GMP (özel iyi üretim uygulamaları dışında imal edilebilmesi olasılığı ortaya) Yerine tesisleri ve personel birden ürünler üretmek için otomatik protokolleri denetleyebilir kan bankacılığı tesislerinde üretilecek.

Bu çalışmada, bir otomatik hücre ilave CCS Sistemi CMV pp65-spesifik T hücrelerinin otomatik üretimi için kullanılmıştır. CMV-spesifik T hücreleri, üç aferez hücre ürünlerinden zenginleştirildi. Kararlı durum aferez ürünü 10 10 toplam nükleer hücreler (TNC) CMV seropozitif donörden 2 saat boyunca hasat ve üretilmiştir. 10 9 TNC CMV pp65 türetilmiş peptidler (60 nmol), 4 saat ve T hücreleri, otomatik hücre zenginleştirme cihazda CCS kullanılarak izole edildi salgıla...

Watch this Scientific Journal Video about Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System at JoVE.com

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

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 ...

automated-cell-enrichment-cytomegalovirus-specific-t-cells-for

Research

JoVE Journal

Immunology and Infection

12.3K Görüntüleme.  U.T. MD Anderson Cancer Center. 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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

İmmünoloji ve Enfeksiyon

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 ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

Primer insan T hücrelerinin piggyBac Transposon Sistem Modifikasyon

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Peptid: Epitop-spesifik T-hücrelerinin MHC tetramer bazlı Zenginleştirme

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

T Hücreleri İçin Kantitatif Yüksek throughput Tek hücreli Sitotoksisite Testi

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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 ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Kullanarak İzleme Dendritik Hücre Göç<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Manyetik Rezonans Görüntüleme

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Fare Naif CD4<sup&gt; +</sup&gt; T Hücre İzolasyonu ve<em&gt; In vitro</em&gt; T hücre alt içine Farklılaşma

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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.