Yazarlar, bu protokolün geliştirilmesi için yardım Jenkins laboratuar Andre Han ve Lawrence Yen teknik yardım ve üyelerine teşekkür etmek istiyorum.

1. Lenfoid Doku gelen Hücre İzolasyonu <ol><li> Bir küçük kare ihtiva eden bir 60 mm kültür çanağı, buz cEHAA (EHAA +% 10 FBS, pen / strep, gentamisin, 2 mM L-glutamin, 55 mM 2-merkaptoetanol) ya da diğer eşdeğer T-hücresi orta 1 ml ekleyin buz üzerinde 100 mikron naylon örgü ve yer. </li><li> Fare Euthanize. </li><li> Dalak ve mümkün olduğunca çok sayıda kolayca erişilebilir lenf düğümleri kaldırın. Bunlar en az inguinal, aksiller, brakial, servikal ve mezenterik lenf düğümleri içermelidir. Kültür çanak naylon örgü üstüne yerleştirin. </li><li> Yavaşça püre lenfositler kurtarmak için naylon örgü üzerinde, kapalı bir 1,5 ml mikrofuge&#39;de tüpünün üst kısmı düz lenfoid doku kullanma. Bir süspansiyon içine hücre çalışması için cEHAA ve pipet aşağı ve yukarı başka bir 1 ml ekleyin. 15 ml polipropilen santrifüj tüpünün üst üzerine yerleştirilen naylon örgü başka bir parça ile hücreleri aktarın. Buz cEHAA, po başka bir 1 ml çanak ve örgü durulayınaynı tüp içine miktarlar oling. 4 ml arasında bir nihai hücre süspansiyonu hacmi elde etmek için tekrarlayın. </li><li> 15 ml&#39;lik bir son hacim için (PBS +% 2 FBS,% 0.05 azid) sıralayıcı soğuk tampon ilave edin ve 4, 300 x g&#39;de 5 dakika boyunca santrifüj tüpü ° C. </li><li> Dikkatli bir şekilde herhangi bir sıvı damlacıkları tüpün iki tarafında kalan emin olarak süpernatant aspire. Yaklaşık olarak iki kat daha pelet kendisi ile eşit bir son hacim Fc blok (sıralayıcı tamponu + 2.4G2 antikor) olarak hücre pelet yeniden süspanse edin. Örneğin, saf bir fare dalak ve lenf düğümleri genellikle yaklaşık 100 ul hücre peleti üretir. Bu durumda, 200 ul hacim getirmek için Fc blok 100 ul ilave edin. Hücre topaklanma büyük ölçüde meydana gelmiş ise, dikkatli bir pipet ucu ile bu noktada cep tutam çıkarın. </li></ol> 2. Tetramer Boyama <ol><li> 10 nM (ya da deneysel olarak optimum konsantrasyon) bir son konsantrasyon için PE-veya APC-etiketli pMHC tetramer ekleyin. </li><li> Mix ve inkübasyonOda sıcaklığında (ya da deneysel olarak optimum süre ve sıcaklığı) 1 saat boyunca te. </li><li> 15 ml&#39;lik bir hacme kadar soğuk tampon sıralayıcı ekleyin ve 300 x g&#39;de, 4 ° C&#39;de 5 dakika süreyle santrifüj tüpü Buz üzerinde veya ° C andan itibaren 4 numune tutun. </li><li> Dikkatli bir şekilde herhangi bir sıvı damlacıkları tüpün iki tarafında kalan emin olarak süpernatant aspire. 200 ul son hacim için sıralayıcı tampon içinde hücre pelet yeniden süspanse edin. Çift tetramer zenginleştirme için, 150 ul nihai hacim için tekrar süspansiyon. </li></ol> 3. Manyetik Zenginleştirme <ol><li> Miltenyi anti-PE ya da anti-APC mikroboncuk 50 ul ekle. Çift tetramer zenginleştirme için, her ikisi de 50 ul ilave edin. </li><li> 4&#39;te karıştırın ve 20 dakika için inkübe ° C </li><li> 15 ml&#39;lik bir hacme kadar soğuk tampon sıralayıcı ekleyin ve 300 x g&#39;de, 4 ° C&#39;de 5 dakika süreyle santrifüj tüpü </li><li> Beklerken, bir MidiMACS veya QuadroMACS mıknatıs bir Miltenyi LS manyetik sütun kurdu. Bir açık bir 15 ml polipropilen santrifüj tüpüne yerleştirinsütununun altında doğrudan rafa. </li><li> O 15 ml tüp içine akabilmesi için, sütunun tepesine sıralayıcı tampon 3 ml ilave edilir. </li><li> Sütunun üstüne bir 100 mikron naylon örgü kare yerleştirin. </li><li> Hücreler iplik bitirdiğinizde, dikkatlice süpernatant aspirat ve sıralayıcı tampon 3 ml pelet tekrar süspansiyon haline getirin. </li><li> Sütunun üst üzerine naylon ağ yoluyla hücre süspansiyonu aktarın. </li><li> Hücre süspansiyonu tamamen sütun içine boşalttıktan sonra, sıralayıcı tampon başka bir 3 ml&#39;lik tüp orijinal durulama ve sütun içine naylon ağ üzerinden aktarmak tampon, süreç içinde ağ durulama. Naylon örgü atın. </li><li> Tampon tamamen sütun içine boşalttıktan sonra, kolona sıralayıcı tampon başka bir 3 ml eklenir. </li><li> 3 x 3 ml yıkama toplam 10 adımı yineleyin. </li><li> Yeni bir 15 ml polipropilen santrifüj tüpü üzerinde mıknatıs ve yerden sütun çıkarın. </li><li> T sıralayıcı tampon 5 ml ekleyino sütun. </li><li> Hemen sütunun üst içine piston takın ve bir sürekli hareket halinde, tüpe kolonu tampon zorlayarak, piston tüm yol aşağı doğru itin. </li><li> 300 x g&#39;de 5 dakika boyunca yıkandı bağlı fraksiyonu ve akan ilişkisiz bir fraksiyonu, 4 ° C&#39;de santrifüje içeren tüpler </li><li> Ayrıca herhangi bir sıvı damlacıkları tüpün iki tarafında kalan emin olarak bağlı fraksiyondan süpernatan dikkatlice aspire. Tam olarak 95 ul son hacim için sıralayıcı tampon içinde hücre pelet yeniden süspanse edin. Aspire ve 2 ml arasında bir nihai hacim için bağlanmamış kısmı tekrar süspansiyon. </li></ol> 4. Akış Sitometri <ol><li> Her bir fraksiyonu için, 5 ul kaldırmak ve bir 5 ml FACS tüp içinde sayım boncuk 200 ul (sıralayıcı tampon içinde 200,000 / ml &#39;lik bir konsantrasyon ile ayarlanmış olarak) eklenir. 4 at kenara ° C analizi için geç. Zamandan tasarruf etmek için, boncuklar deney başlamadan önce etiketli tüpler önceden aliquoted edilebilir. </li><li> Ben hazırlayınAntikorların aster karışımı hücreleri yüzey işaretleyicileri (Tablo 1) leke. Zamandan tasarruf etmek için, bu deney başlamadan önce yapılabilir. </li><li> Bağlı fraksiyonu için, tüp içinde hücrelerin ~ 90 ul doğrudan antikor kokteylinin bir doz ekleyin. Bağsız fraksiyonunun analiz istenirse, bir 5 ml FACS tüp için hücre 90 ul aktarımı ve antikor kokteyli bir doz ekleyin. </li><li> Akış sitometresi için tek renk telafisi denetimlerin paneli yukarı ayarlayın. Kullanılmak üzere her florokrom için, karşılık gelen florokrom ile konjuge anti-CD4 antikoru ile bir 1 ul FACS 5 ml tüp içinde kalan bağsız fraksiyonu hücreleri 50 ul ilave edilir. Bunun yanı sıra bir boyanmamış kontrol ayarlamayı unutmayın. </li><li> Girdap ve 4, 30 dakika için tüm örnekler inkübe ° C </li><li> 300 xg 5 dak, 4 ° C &#39;için her bir tüp ve santrifüj ile sıralayıcı tampon 5 mL </li><li> Bağlı fraksiyonu için, tampon dikkatlice sıralayıcı 200 ul süpernatan aspire hücreleri ve tekrar süspansiyonfer. 1.2 ml FACS mikrotüp içine hücreleri aktarın. Aynı mikrotüp içine sıralayıcı tampon ve havuz başka bir 200 ul ile tüp durulayın. Hücre kümeleri açık ise, bir 50 um filtre aracılığıyla hücre geçmektedir. </li><li> Ilişkisiz fraksiyonu ve tazminat kontrolleri, süzün veya supernatant ve tekrar süspansiyon 2 ml sıralayıcı tampon aspire için. </li><li> Tek renk telafisi kontrollerini kullanarak akış sitometresi ayarlayın. Kaydedilecek ek bir parametre olarak yan dağılım genişliği (SSC-W) seçin. </li><li> Şekiller 1 ve 2 de gösterildiği gibi, + ya da CD8 + T hücreleri CD4 belirlemek için ardışık içerme bir geçitlerinin dizisi kullanılarak boyanan numuneler analiz edin. Bağlı kesirler için, yukarı 2.000.000 toplam etkinlik azami mümkün olduğunca çok sayıda hücreleri toplamak. Ilişkisiz kesirler için, 1.000.000 toplam etkinlik toplamak. Alma hızı saniyede etkinliklerde veya 3000 altında tutun. </li><li> Aynı makine ayarları kullanarak, sayma boncuk örnekleri analiz. 10,000 toplam etkinlik toplayın. &lt;/ Li&gt; <li> FCS dosyaları gibi tüm verileri kaydedin. </li></ol> 5. Veri Analizi <ol><li> FlowJo yazılımı kullanarak sayma boncuk örnekleri için FCS veri dosyalarını analiz edin. Plot ileri FITC ile saçılım ve (Şekil 1 ve 2) sayma boncuklar Kottbusser bir kapı ayarlayın. Her bir numune içinde tespit edilen boncuk sayma sayısını belirler. Toplanan hücre olayların sayısını belirlemek için toplanan olayların toplam sayıdan çıkart. </li><li> Kutu 1&#39;de denklemler kullanılarak her bir örnek tüm hücrelerinin toplam sayısını hesaplayın. </li><li> Lekeli ilişkili ve ilişkisiz fraksiyonu örnekleri için FCS veri dosyalarını analiz edin. Lenfoid +, yan genişlik dağılım-lo, dökümü-, CD3 +, CD4 + ya da CD8 +, tetramer Şekiller 1 ve 2 de gösterildiği gibi, her bir numune içinde + T hücreleridir. Belirlemek için ardışık içerme kapıları bir dizi ayarlama </li><li> CD4 tanımlamak için kullanılan içerme kapılarının her biri sıklığı + tetramer göre örnek bir hücre toplam sayısı çarpma+ Ya da CD8 + tetramer + hücreleri epitopu özgü T hücreleri (Kutu 1) toplam sayısını hesaplamak için kullanılır. </li></ol>

<ol>
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Çıkar çatışması ilan etti.

Bu protokol tarafından sunulan pMHC tetramer tabanlı cep zenginleştirme yöntemi endojen T hücre çizgilerinden epitop-spesifik T hücreleri incelemek için güçlü bir araçtır. PMHC tetramerleri kullanımı, doğrudan doğruya aynı kökten gelen pMHC ligandlar bağlamak için kendi TCRS yeteneğine dayalı epitopa spesifik T hücrelerin saptanması sağlar. Zenginleştirme antijen-spesifik T hücrelerinin çok nadir topluluğunun, genetik ya da haberci frekans herhangi bir düzenlemesi olmadan T hücrelerinin e...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Reaktif</strong</td&gt;<td<strong&gt; Bayilik</strong</td&gt;<td<strong&gt; Katalog numarası</strong</td&gt;</tr&gt;<tr&gt;<td&gt; PE veya APC konjuge pMHC tetramer (veya multimer)</td&gt;<td&gt; NIH tetramer çekirdek elde, araştırmacı tarafından yapılmış, ya da ticari kaynaklardan satın</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-PE konjuge manyetik mikroboncukları</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-048-801</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-APC konjuge manyetik mikroboncukları</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-090-855</td&gt;</tr&gt;<tr&gt;<td&gt; LS manyetik sütunlar</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-401</td&gt;</tr&gt;<tr&gt;<td&gt; MidiMACS veya QuadroMACS mıknatıs</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-302 veya 130-090-976</td&gt;</tr&gt;<tr&gt;<td&gt; Hücre sayımı boncuk</td&gt;<td&gt; Yaşam Teknolojileri</td&gt;<td&gt; PCB-100</td&gt;</tr&gt;</tbody&gt;</table

peptide:mhc tetramer-based enrichment of epitope-specific t cells

Vivo deneysel modellerde olan adaptif bağışıklık inceleyen araştırmacılar için temel bir gereklilik kendi T hücre antijen reseptör (TCR) özgüllük esas T hücreleri belirlemek için bir yetenek. Pek çok dolaylı yöntemlerle T-hücrelerinin bir yığın nüfus belirli bir antijen ile epitopu özgü T hücreleri proliferasyonu, sitokin üretimi, ya da aktivasyonu 1 ifadesi olarak işlevsel bir yanıt ölçümü ile tespit edilir ile in vitro uyarılmış olduğu kullanılabilir. Bununla birlikte, bu yöntem sadece birçok olası fonksiyon gösteren bir epitopuna özgü T hücreleri belirlemek, ve saf haberci frekanslarda epitopa spesifik T hücreleri saptamak için yeterli değildir. Popüler bir alternatif, TCR transjenik adoptif aktarım modeli olan bir TCR transjenik fare monoklonal T hücreleri EASI olabilir epitopa spesifik T-hücrelerinin geniş bir haberci nüfus oluşturmak için histocompatible taşıyıcılara yerleştirildiği tohumlanmıştırly bir congenic işaretleyici antikor 2,3 kullanımı ile izlenir. Güçlü olmakla birlikte, bu yöntem, 4,5, tek bir epitopu için spesifikliğe sahip olan T hücrelerinin unphysiological frekansı ile ilişkili deneysel eser uğrar. Ayrıca, bu sistem, bir poliklonal popülasyon içindeki epitopa spesifik T hücre klonlarının fonksiyonel heterojen araştırmak için kullanılamaz. MHC (pMHC) kompleksleri: kazanılmış bağışıklık incelemek için ideal bir şekilde sadece kendi akraba peptid bağlayıcı ile TCR özgüllük ayıran bir yöntem kullanılarak, endojen T-hücresi epitopu repertuarından-spesifik T hücrelerinin doğrudan saptanması içermelidir. PMHC tetramerleri ve akış sitometrisi kullanımı bu 6 gerçekleştirir, ancak şu antijen kaynaklı klonal büyüme bulunamadı epitopa spesifik T-hücrelerinin yüksek frekans popülasyonlarının tespiti ile sınırlıdır. Bu protokol, bir pMHC tetramerleri kullanımı ile koordine yöntem ve magnezyum tanımlamaktik hücre zenginleştirme teknoloji fare lenf dokuları 3,7 son derece alçak frekans epitopa spesifik T hücrelerin saptanması sağlamaktır. Bu teknik sayesinde, bir kapsamlı immün yanıtın tüm aşamalarında farelerde endojen T hücrelerinin tamamı epitop özel popülasyonlarda izleyebilirsiniz.

Şekil 2&#39;de daha önceden ilgili peptid + CFA ile bağışık hale getirilen farelerin için temsilci veriler göstermektedir iken Şekil 1, saf farelerden alınan pMHCII tetramer zenginleştirilmiş dalak ve lenf düğümü numuneleri için temsili akış sitometrisi parselleri göstermektedir. Seri yolluk CD4 analizi + T hücre popülasyonlarının gelen autofluorescent ve diğer istenmeyen olaylar kaldırır. CD8 + T hücre populasyonunun CD4 pMHCII tetramer boyama + T hücreleri...

Watch this Scientific Journal Video about Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells at JoVE.com

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

Epitopa spesifik T-hücrelerinin alçak frekans popülasyonları izole etmek ve akış sitometrisi ile analiz etmek için bunları MHC tetramerleri ve manyetik mikro-küreler: Bu protokol peptit kullanımı açıklanmıştır. Bu yöntem ilgi endojen T hücre popülasyonlarının doğrudan çalışma sağlar<em&gt; In vivo</em&gt; Deneysel sistemler.

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 ...

peptidemhc-tetramer-based-enrichment-of-epitope-specific-t-cells

Research

JoVE Journal

Immunology and Infection

17.8K Görüntüleme.  Massachusetts General Hospital and Harvard Medical School. Epitopa spesifik T-hücrelerinin alçak frekans popülasyonları izole etmek ve akış sitometrisi ile analiz etmek için bunları MHC tetramerleri ve manyetik mikro-küreler: Bu protokol peptit kullanımı açıklanmıştır. Bu yöntem ilgi endojen T hücre popülasyonlarının doğrudan çalışma sağlar In vivo Deneysel sistemler.

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

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

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 ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T hücrelerinin türetilmesi<em&gt; İn Vitro</em&gt; Fare Embriyonik Kök Hücreleri

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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.

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 ...

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

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

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

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

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

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T hücreleri dendritik hücreler gelen Transinfection tarafından Bakteriler Yakalama

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

İnsan CRISPR-Engineered CAR-T Hücrelerinin Üretimi

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Adoptive Transfer of P14 T-Cells into Tumor-Bearing Mice

P14 T-Hücrelerinin Tümör Taşıyan Farelere Evlat Edinilerek Transferi

Fare Akciğerlerinde Antijene Özgü T Hücrelerinin İzolasyonu ve Karakterizasyonu

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Yazar Spotlight: Tetramerler Kullanarak Fare Akciğerlerinde Antijene Özgü T Hücrelerini Tespit Etmek için Optimize Edilmiş Protokol

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

Fare Akciğerlerinden Nadir Antijene Özgü T Hücrelerinin Peptit ile Tanımlanması: Majör Histouyumluluk Kompleksi Tetramerleri

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...