The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

NOT: Bu protokol, insan gönüllülerden periferal kan örneklerinin kullanılmasını gerektirir. Insan denekler ile tüm araştırma gözden ve Helsinki Bildirgesi (2013) ve Sağlık Sigortası Taşınabilirliği ve 1996 Sorumluluk Yasası ile uyumu sağlamak üzere bir İnsan Araştırmaları Kurumsal Değerlendirme Kurulu tarafından onaylanmış olması gerekir. Tüm kan T-hücrelerinin 1. izolasyonu <ol><li> Başlamadan önce, oda sıcaklığına kadar yoğunluk gradyan orta ısıtın. Kısım 2-4 steril 15 ml konik santrifüj tüplerine yoğunluk gradyan ortamının 4 ml (1 tüp her biri, 10 ml seyreltilmiş kan hacmi için kullanılacaktır). </li><li> 1-2 sodyum heparin sprey kan 10-20 ml kaplı (yeşil-üst) venöz kan alma tüpleri edinin. Eğitimli bireylerin gözetiminde insan numuneleri toplamak ve Kurumsal Değerlendirme Kuruluna göre protokolünü onayladı. </li><li> % 70 etanol ile, tüplerin dış silin. Dikkatle fi üstleri kaldırmaktoplama tüpleri doldurulmayan, steril bir 50 ml santrifüj tüpüne kan süzün. </li><li> Her bir kan toplama tüpüne 10 ml steril fosfat tamponlu tuz (PBS) ekleyin. , PBS toplamak decanted tam kan eklemek ve hafifçe karıştırın. </li><li> 25 ul insan T hücresi zenginleştirme Kokteyl / 2 mL toplam hacim. 20 dakika oda sıcaklığında inkübe edin. </li><li> Yavaşça yoğunluk gradyan ortamının üstte 1 seyreltilmiş kan: 10 ml&#39;lik bir pipet kullanılarak, 1, 10 ml kadar katman. Yoğunluk gradyan ortamın yüzeyini bozmak için dikkatli olun. </li><li> 20 o C&#39;de santrifüj katmanlı kan ve yoğunluk gradyan orta 20 dakika boyunca 1200 x g&#39;de </li><li> Yoğunluk gradyan orta ve yoğunluk gradyan orta ve seyreltilmiş plazma arasındaki ara yüzde lökosit tabakası arasındaki ara yüzü bozmayacak dikkat ederek, santrifüj tüpleri çıkarın. 5 ml&#39;lik bir pipet kullanarak dikkatli bir şekilde lökosit faz toplanır ve yeni bir steril 50 ml konik küvet transferör. </li><li> 50 ml toplanan PBL hacmini getirmek ve hafifçe karıştırmak için PBS ekleyin. </li><li> 20 ° C&#39;de 5 dakika boyunca 600 x g&#39;de santrifüj Durusu. </li><li> Süspanse tamponu (steril filtre edilmiş PBS% 1 BSA ihtiva eden) ayırma sitometrisi 10 ml&#39;lik steril bir akış hücreleri topak. Örnek hücre süspansiyonu, 10 ul, 1:10 seyreltilmiş bir hemositometre kullanılarak hücre sayısı tripan mavisi (90 ul ekle) (beklenen verim 1-4 x 10 6 hücre tam kan / tüp). <ol><li> 5 ml&#39;lik bir tüpe transfer 1 ml lik bir çıkarın ve tetramer etiketli olmayan T hücrelerinin analizi için buz üzerinde tutulur. Buz üzerinde hücre süspansiyonu tutun. </li></ol></li></ol> Alloantijen-spesifik T hücrelerinin 2. Manyetik zenginleştirilmesi <ol><li> 1 Alloantijen pMHC tetramerini sulandırmak: 100 steril sıralama tamponunda. </li><li> Santrifüj Hücre süspansiyonu, 20 ° C&#39;de 5 dakika boyunca 600 x g&#39;de (adım 1.11 9 mi) Durusu. </li><li> Peletlendi için pMHC tetramer Alloantijen seyreltilmiş 50 ul ekleyinHücreler (10 7 hücreleri kadar). Nazik pipetleme karıştırın. Steril bir 5 ml tüp aktarın. Oda sıcaklığında 30 dakika boyunca inkübe edin. </li><li> Sıralama tampon 2 ml ekleyin. 20 ° C&#39;de 5 dakika boyunca 600 x g&#39;de santrifüj Durusu. </li><li> 100 ul sıralama tamponunda süspanse hücreler. 10 ul Biotin seçimi kokteyl ilave edilir ve oda sıcaklığında 15 dakika inkübe edilir. </li><li> 5 ul manyetik nanopartikülleri ilave edilir ve oda sıcaklığında 10 dakika inkübe edilir. </li><li> 2.5 ml sıralama tampon ve pipetleme hafifçe karıştırın ekleyin. 5 ml tüp aktarmak, 100 ul kısım çıkarın ve ön zenginleştirme hücrelerin analizi için buz üzerinde tutmak. </li><li> Hücre ayrılma mıknatıs hücre süspansiyonu ihtiva eden 5 ml&#39;lik tüp yerleştirin. Bu kapak, tüpün üstüne gevşek olması gerekmektedir. Oda sıcaklığında 5 dakika boyunca inkübe edin. </li><li> Yavaşça tüpten kapağı çıkarın. Birlikte tüp ve mıknatıs tutma, taze 5 ml tüp içine tüp içeriğini süzün. Dokunun veya tüp içeriğini sallamayınsıvının son damla çıkarın. </li><li> Mıknatıstan tüpü çıkarın. 2 ml soğuk sıralama tampon ekleyin ve pipetle hafifçe karıştırın. 2, bir hemositometre kullanılarak hücrelerin sayısı tripan mavisi (10 ul ekle): Örnek hücre süspansiyonu, 10 ul, 1 seyreltin. </li></ol> Tek hücreli Sitometrisi hücre sıralama için T hücrelerinin 3. hazırlanması <ol><li> Hücrelerin tüm tüplere soğuk sıralama tampon ekleme (tetramer etiketlenmemiş, tetramer etiketli zenginleştirilmiş un, tetramer ve etiketli zenginleştirilmiş) 3 ml hacim getirmek. </li><li> 4 o C&#39;de 5 dakika boyunca 600 xg&#39;de Santrifüj ve decant tampon hücreleri kurtarmak için. </li><li> 25 ul soğuk tür tampon pelet hücreleri tekrar süspansiyon. 5 ul insan Fc blok ekleyin. 20 dakika boyunca buz üzerinde inkübe edin. </li><li> Akış sitometri sıralama için antikorlar hazırlayın. 15 ul sıralama tampon, 15 ul anti-CD5, 15 ul anti-CD14, ve 15 ul anti-CD19 karıştırın. </li><li> Hücrelerin her biri tüp antikor karışımı 20 ul ekle. 20 dakika boyunca buz üzerinde inkübe edin. </li><li> Her tüpe soğuk çeşit tampon 2 ml ekleyin. </li><li> 4 o C&#39;de 5 dakika boyunca 600 xg&#39;de Santrifüj ve decant tampon hücreleri kurtarmak için. </li><li> Sıralama tampon maddesi içinde 1 -2 x 10 6 hücre / ml&#39;lik bir konsantrasyonda tekrar süspansiyon hücreleri. </li><li> Insan T hücre kültür ortamının kısım 100 ul (Iscove DMEM, 2 mM Glutamax, 10 mM HEPES, 50 ug / ml gentamisin, 50 uM 2-merkaptoetanol,% 10 ısı ile inaktive edilmiş insan AB serumu, ve 2.5 ng / ml rekombinant insan ile desteklenmiş IL-2), 96 oyuklu tabanı yuvarlak hücre kültürü plakasının her oyuğuna. Buz üzerinde tutun. </li></ol> Tek hücreli Sitometrisi Sorting tarafından Tetramer-etiketli T hücrelerinin 4. İzolasyonu <ol><li> Alloantijen pMHC tetrameri etiketli T hücreleri (Şekil 2.a) tanımlamak için yolluk parametrelerin akım sitometri kurmak. B hücreleri ve CD14 ifade CD19 hariç, lenfositlerin ikilileri, kapıyı ortadan kaldırmak için stratejiler yolluk kurmak için ön zenginleştirme tetrameri etiketli kısmını kullanınmonositler ifade ve CD5 ifadesi ile, T hücreleri tanımlar. (Tetramer etiketli olmayan örnekten hücreler bu kapıdan giren ve - - CD19, CD5 + CD14 0%) ile tetramer bağlanması T hücrelerin belirlenmesine yolluk kurmak için bir floresan gibi tetrameri eksi bir kumanda ile işaretli olmayan örnek kullanın . Not: aşırı kısıtlayıcı yolluk stratejileri izole edilen T hücrelerinin sayısını azaltırken, antijene özel niteliği olmayan, T hücreleri veya T hücrelerinin izolasyonu ile sonuçlanır yeterince sıkı değildir stratejileri yolluk. </li><li> Tek hücre sıralama için plaka parametreleri programlamak. CD19 - - Her kuyuda tetramerini + hücreyi 1 CD5 + CD14 yerleştirmek için sıralayıcı yönlendirin. Her satırda (Şekil 2.S iyi (hücreler - CD19 - - tetrameri 100 CD5 + CD14) de 1 negatif kontrol (hücreleri kuyunun içine sıralanır) ve 1 pozitif kontrol içerecek şekilde ayarlanan plakayı yönlendirin </strong&gt;). </li><li> Ayırıcı stratejisi ve plaka kurulumu sitometrisi düzenlenen akım kullanarak, doğrudan bir akış sitometrik hücre ayırıcıyla 96 oyuklu bir plakaya bireysel tetramer bağlanması T hücreleri izole etmek. </li></ol> 5. Kültür ve alloantijen-özgü T hücresi klonlarının Genişleme <ol><li> 20 ° C&#39;de, hücre sınıflandırılması; 5 dakika boyunca 600 x g&#39;de santrifüj plaka tamamlanmasından sonra, % 6 CO 2 inkübatör 37 o C yerleştirin kültür plakası. </li><li> 30 saniye vorteksleyin uyarıcı, anti-CD3 / anti-CD28 mikro boncuklar boncuklar tekrar süspansiyon. </li><li> Toplanan T hücrelerinin uyarılması için gerekli olan aktivatör boncuk hacmi (aktivatörünün mikro-/ oyuk 0.5 ul) hesaplayın. Steril bir 5 ml tüp uyarıcısı boncuk hesaplanan hacim aktarın. 1 ml insan T hücre kültür ortamı ve vorteks ekleyin. </li><li> Mıknatıs microbead süspansiyonu ile tüp yerleştirin. Bu kapak, tüpün üstüne gevşek olması gerekmektedir. Oda sıcaklığında 2 dakika süreyle inkübe edin. </li><li> Yavaşça çıkarıntüpten kabı. Taze 5 ml tüp içine, birlikte decant tüp içeriği tüp ve mıknatıs tutma. Dokunun veya tüp içeriğini sallamayın. </li><li> Mıknatıstan tüpü çıkarın. Insan T hücre kültür ortamı ilave edin (mikrotaneciklerin, 100 ul ortam / 0.5 ul, başlangıçta ilave edildi). </li><li> Kısım 96 oyuklu plakanın her oyuğuna aktivatör boncuk süspansiyonu 100 ul. % 6 CO 2 inkübatör 37 o C kültür plaka inkübe. </li><li> Mikroskobik inceleme ile günlük hücre büyümesini izleyin. 7 günlük bir kültürden (Şekil 3a) - çoğalan hücrelerin küçük kümeler 5 sonra mikroskopla gözlenebilir. Not: bu noktada T hücresi kültürlerinin Görselleştirme zor olabilir, ve bu aşamada gözlemlenebilir hücre kümelerinin olmaması artan bir T hücresi eksikliği olmayabilir. </li><li> Hücre izolasyonu 14 gün sonra, dikkatli bir şekilde kültür üst kapalı ve ortam 100 ul çıkararak besleme kültürleri 100 μ ile değiştirinL, taze insan T hücre kültürü ortamı. % 6 CO 2 inkübatör 37 o C kültür plaka kuluçkaya devam edin. </li><li> Mikroskopik inceleme ve kültür ortamının rengi değerlendirilmesi günlük hücre büyümesini izlemek. 3 gün orta değişiklikten sonra - Büyük hücre kümeleri mikroskobik 2 görünür olmalıdır. Makroskobik olarak, hücre topakları ortam değişikliği takiben 14 gün boyunca görünür hale gelmelidir. </li><li> Hücre izolasyonunu takiben 28 gün, mikroskobik inceleme ile büyüyen klonları tanımlamak. 200 ul insan T hücre kültür ortamı içeren 48-çukurlu bir doku kültür plakasının her bir gözeneğine büyüme pozitif 96 oyuklu plaka kültürlerin 200 ul hacim aktarın. </li><li> Uyarıcı, anti-CD3 / anti-CD28 mikro boncuklar 12.5 ul içeren 100 ul ekle (- 5.6 bölümlerde 5.2 &#39;de tarif edildiği gibi hazırlanmıştır). % 6 CO 2 inkübatör 37 o C kültür plaka inkübe. </li><li> Mikroskobik eski tarafından günlük kültür büyümesini izlemekaminasyon ve kültür ortamının rengi değerlendirilmesi. T hücresi kültürü&gt; 2x10 6 / ml&#39;ye ulaştığı zaman (tipik olarak 3-5 gün tekrar uyarma işleminden sonra, ortam başladığı gün ile tahmin edilebilir saman-sarı renk), 24 oyuklu doku kültürü plakası, bir oyuğuna transfer kültürü ve 500 ul, insan T hücre ortamı ilave edin. </li><li> 10-14 gün pMHC tetramerinin analizi bağlama (bölüm 3 ve 4.1 açıklanan etiketleme ve yolluk stratejisini kullanarak) flow sitometri ile Alloantijen özelliğinin değerlendirilmesi T hücre kültürünün 200 ul toplamak, yeniden uyarma izleyin. </li></ol> 6. Uzun süreli yeniden uyarma ve Kültür T hücresi klonlarının <ol><li> Sürekli her 14 günde bir olabilir, arzu edilen özellikteki klonal T hücresi kültürlerini yeniden canlandırmak ve genişletmek. Son anti-CD3 / CD28 mikroboncuk uyarımını takiben 14. günde, steril bir 5 ml&#39;lik bir tüp içine T hücresi kültürlerini toplayın. 2,5 ml&#39;lik bir hacme, tek tüp içinde, aynı T hücresi klonunun birden fazla kuyu birleştirin. </li&gt; <li> 2.5 ml nihai hacme getirmek ve pipetleme hafifçe karıştırmak için orta ekleyin. </li><li> Kültürden eski, mikro çıkarmak için bir hücre ayırma mıknatıs T hücresi kültürü içeren 5 ml&#39;lik tüp yerleştirin. Bu kapak, tüpün üstüne gevşek olması gerekmektedir. Oda sıcaklığında 5 dakika boyunca inkübe edin. </li><li> Yavaşça tüpten kapağı çıkarın. Birlikte tüp ve mıknatıs tutma, taze 5 ml&#39;lik bir tüp içine hücre süspansiyonu süzün. Dokunun ya da sıvının son damla kaldırmak için tüp içeriği sallamayın. </li><li> 4 ml nihai hacmi getirmek için orta ekleyin. Örnek 10 ul Hemasitometre kullanarak hücreleri saymak için. </li><li> Oda sıcaklığında 5 dakika boyunca 600 x g&#39;de santrifüj ile T hücrelerinin yeniden elde etmek. </li><li> Durusu süpernatant. 10 6 hücre / ml insan T hücre kültürü ortamında yeniden süspanse T hücreleri. Aktarım 1, 24 oyuklu doku kültür plakasının her bir oyuğuna ml&#39;lik eş payları. </li><li> Uyarıcı, anti-CD3 / a 12.5 ul ihtiva eden 100 ul ortam eklenerek T hücrelerini uyarmak YenidenYüzgeç CD28 microbeads (- 5.6 gibi bölümlere 5.2 açıklanmıştır). % 6 CO 2 inkübatör 37 o C&#39;de inkübe edilir. </li></ol>

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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

T lenfositleri adaptif bağışıklık sisteminin önemli bileşenleridir. T hücreleri, sadece doğrudan değil, efektör çeşitli mekanizmalar aracılığıyla patojenlere karşı koruyucu bir immün yanıtlara aracılık değil, aynı zamanda aktif kendini immünolojik toleransı muhafaza edilmesi ve bağışıklık sisteminin diğer hücrelerinin tepkileri yönlendirilmesi için sorumludur. Bu işlevler, bir T hücresi reseptörü (TCR) bağlanması, sitokinler ve kemokinlerin ve metabolitleri dahil olmak üzere 1, entegre sinyalleri, bir dizi içine yönlendirilir. Adaptif bağışıklık T hücre rolü tanımlayan karakteristik spesifisite sağlamaktadır Bu sinyaller arasında, TCR, özel bir önem taşır. TCR, T hücre efektör işlevi başlatmak sinyallerini temin etmek üzere bir çok özel ve hassas bir şekilde MHC (insan ortologu HLA) molekülleri (pMHC kompleksleri) tarafından sunulan lineer peptid antijen ile etkileşime girer. PMHC ligandlar ile TCR etkileşimleri biyokimyasal parametreleri T özgüllük sadece sağlarHücre aktivasyonu, aynı zamanda daha sonra T-hücresi işlevinin 2 niteliksel etkisi vardır. Bu nedenle, eğitim T hücre fonksiyon, genellikle tanımlanmış antijenik özgünlüğe sahip klonal T hücrelerinin tepkileri incelenmesi gerekir. 10 8 farklı αβTCRs 3-4 - yaklaşık 10 ila 12 αβ hücresi içeren insan T hücresi bölmesi, tahminen 10 7 içerir. Bu farklı repertuar koruyucu bağışıklık için bir T hücre yanıtını gerektirecek potansiyel patojenlerin peptidlerin geniş bir dizi tanınması için bir fırsat sunuyor. Bu antijene 5 önce bağışıklık yanıtının yokluğunda 10 -7 - kendi kendine MHC tarafından sunulan belirli bir yabancı antijene yanıt olarak T hücrelerinin frekansı 10 -4 mertebesinde olduğu tahmin edilmektedir. Naif T hücre repertuar peptid antijenleri ve sınır tepki gösteren öz-MHC tanıma yeteneğini sağlamak için timus seçimi ile şekillenmektedirkendinden peptit antijenlerine karşı ivity koruyucu bağışıklığı 2 aracılık potansiyel kullanımını maksimize edilmesidir. Bu ihlali reaktifliği, göreceli olarak büyük bir frekans, 10 -3 tasarlanmış Bununla birlikte, - İmmünolojik olarak naif bulunan bireylerden alınan T hücrelerinin, 10 -4, yabancı MHC moleküllerini hem de onlar mevcut endojen peptidler hem de göz önünde bulundurarak, farklı genetik yapıdaki hücreler ile uyarılmaya yanıt 6. Allojenik pMHC ligandların tanınması, bireysel MHC tarafından sunulan yabancı antigenlerin tanınmasına yapısal olarak benzer olduğu; TCR, allojenik MHC molekülü hem de sunulan peptit 7 her iki kritik biyokimyasal etkileşimleri yapar. PMHC çeşitliliğine allojeneik stimülasyonu sonucunda T hücrelerinin yanıtı sağlam yapısı farklı genetik yapıdaki hücreler, 8 yüzeyi üzerinde mevcut olan kompleksler. Bu, her bir MHC yaklaşık 2 x 10 4 farklı endojen peptit antijenlerini 9 sunulur olduğu tahmin edilmektedir. Bu bAllojenik stimülasyona yanıt readth T hücre alo-tepkiselliği sonuçlanan, host hastalığı (GVHD) alograft reddi veya doku yaması gibi klinik olarak ilgili patolojisi arasında önemli bir yönüdür. Insan T hücresi alloreaktif yanıtlarının Çalışma, geleneksel olarak allojenik hücreleri ile stimüle edilmesini müteakkip bir hassas T hücreleri poliklonal tepkilerin test üzerine yaslanmaktadır. Sınırlayıcı seyreltme ile birlikte aynı allojeneik hücre hattı ile tekrarlı uyarım allojenik HLA 10 tanımlanan tanınması klonal T hücrelerinin üretme yeteneğine sahiptir analiz eder. Endojen pMHC en büyük ve farklı bir repertuar HLA T-hücrelerinin geniş bir repertuar uyaran belirli bir alogeneik kapsamayabilir kompleksleri Ancak, bu yaklaşım tek tek allojenik pMHC ligandlarına yanıtları incelenmesi için sorunludur. Bu toplu popülasyon uyarılması ve sınırlayıcı seyreltme yaklaşım arzu edilen Reacti T hücreleri izole etmek için klonların çok sayıda tarama gerektirecektirTek pMHC bağına karşı vite. Buna ek olarak, tek bir allojenik pMHC liganda yanıt T hücrelerinin frekansı, belirli bir antijene tepki veren, insan T hücresi klonlarının verimli üretimi için bir engel sunulur naif T hücresi popülasyonları arasında nispeten düşüktür. Tanımlama ve poliklonal popülasyonlarından antijen-spesifik T hücrelerinin elde edilmesi fluoroforla işaretlenmiş pMHC multimerlerinin 11 geliştirilmesi ile sağlanmıştır. Bu yaklaşım, bir streptavidin-işaretli flüorofor bağlanarak etiketli rekombinant çözünür biyotinile MHC molekülleri, yüklenen özel peptit antijenlerini kullanmaktadır. PMHC bir multimerizasyon çözünür pMHC ligandlar için TCR&#39;nin bünyesinde düşük bir düzeyde (uM) bir afinite telafi avidite artırır. Etiketlenmiş hücreler tanımlanır ve akış sitometrisi ile izole edilebilir. Bununla birlikte, bu yaklaşım, halen tipik olarak naif T hücresi popülasyonları arasında, antijen-spesifik T hücrelerin, en düşük frekans ile sınırlıdırEn akış sitometrelerinde doğru tanımlama ve tayin sınırından daha az büyüklükte siparişleri. Bu sınırlama gidermek için, tetramer-etiketli hücreler için pMHC tetramer etiketleme ve daha sonra manyetik boncuk zenginleştirmek için bir yöntem, 12 geliştirilmiştir. Bu yöntem, güvenli bir tespit numaralandırma ve düşük frekanslı antijen-spesifik T hücrelerinin elde edilmesi göstermiştir. Bu protokol, spesifik olarak bireysel allojenik pMHC-ligantlarına tepki veren, insan T hücresi klonlarının oluşturulması için etkili bir protokolü tarif eder. Bu protokol, tek kriteri hücrelerinden T hücresi klonlarının üretimini (etkinleştirmeniz sıralama hücre bağlanımını akış sitometrisiyle ve insan T hücrelerinin in vitro kültürü için güçlü bir yöntemle Alloantijen özgü insan T hücrelerinin izolasyonu için multimer, etiketleme ve zenginleştirme pMHC (HLA) &#39;nin uygulandığı Şekil 1&#39;de genel bakış).

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Sodium heparin venous blood collection tube 16 x 100 mm</td>
			<td style="width:223px;">Becton, Dickenson and Company</td>
			<td style="width:119px;">366480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Lymphoprep</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">7801</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:273px;">Rosette Sep Human T Cell Enrichment Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">15061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dulbecco&#39;s PBS, 1x without Ca or Mg</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fluorophore-labeled pMHC tetramer</td>
			<td style="width:223px;">NIH Tetramer Facility</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Biotin Selection Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Selection magnet</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:273px;">TruStain FcX Human Fc blocking solution</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">422301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD5 PE-Cy7 (clone UCHT2)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">300621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD14 FITC (clone HCD14)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">325603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD19 FITC (clone HIB19)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">302205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Iscove&#39;s DMEM, without b-ME or L-glutamine</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">15-016-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin sulfate (50 mg/ml)</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">GT-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human AB serum, male donor</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">HS-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2</td>
			<td style="width:223px;">Peprotech</td>
			<td style="width:119px;">AF 200-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Dynabeads Human T-Activator CD3/CD28</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">11131D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Media</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cell sorting buffer</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS, pH 7.4</td>
			<td style="width:223px;">1 L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BSA</td>
			<td style="width:223px;">10g</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA (0.5 M)</td>
			<td style="width:223px;">2 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human T Cell Culture Medium</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Iscove&#39;s DMEM</td>
			<td style="width:223px;">351.6 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Heat-inactivated human AB serum</td>
			<td style="width:223px;">40 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES (1 M)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax (100 x)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin (50 mg/ml)</td>
			<td style="width:223px;">0.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-mercaptoethanol (14.3 M)</td>
			<td style="width:223px;">1.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2 (1 mg/ml)</td>
			<td style="width:223px;">1 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human alloantigen-specific t cells from peripheral blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

Strateji sıralama Akış sitometrisi, bir manyetik tanecik zenginleştirme ve tek hücreli akışı aracılığı ile özgüllük Alloantijen tanımlanmış olan bu protokol, klonal insan T hücre kültürlerinin üretimini tarif eder. 1, sürecin bir özeti verilmektedir Şekil. <img alt="Şekil 1," src="/files/ftp_upload/52257/52257fig1highres.jpg" /> Şekil 1: Protokol genel protokolü, burada a...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K Görüntüleme.  University of California, San Diego. This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Video: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Genişleme, Saflaştırılması, ve İnsan Periferik Kan NK Hücrelerinin Fonksiyonel Değerlendirme

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

İmmünoloji ve Enfeksiyon

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Co-uyarıcı Sinyal Abluka Alloantigen Uyarım İnsan periferik kan mononükleer hücreleri içinde Alloantigen özel anerji indüksiyonu

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Zoledronate İnsan Periferik Kan Genişleme γδ T Hücreler

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Kanda dolaşan nadir antijen spesifik B hücreleri Discriminative insan monoklonal antikorların üretimi

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Periferik kan ve lenf dokusu birincil insan T hücre sinaptik arabirimi değerlendirilmesi

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Flotasyon Bazlı T Hücresi İzolasyonu, Aktivasyonu ve Mikrokabarcıklar Kullanılarak İnsan Periferik Kan Mononükleer Hücre Örneklerinden Genişleme

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Fonksiyonel Osteoklastların İnsan Periferik Kanı CD14+ Monositlerinden Farklılaşması

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...