Bu çalışma NIH (5U01CA137070) hibe ve Amerikan Kanser Derneği (RSG-09-070-01-LIB), Kanser Araştırma Enstitüsü Araştırmacı Ödülü (MK), Amerikan Kalp Derneği, ön-doktora bursu (KM) ve desteklenmiştir. , Eğitim ve Bilim Bursu (APG) bir İspanyol Bakanlığı. Biz protokol yararlı önerileri için teşekkür Dr. Nicholas Restifo ve Dr. Zhiya Yu (NIH / NCI) istiyorum.

1. Retroviral Construct hazırlayın <ol><li> Alt klon retroviral bir vektör içine ilgi T-hücre reseptör (TCR) geni (Şekil 1, 7 PMSG örnek vektörler, pMIGII 5,8, Cellbiolabs gelen pMXs). TCR α ve β zincir gen eşit ifade sağlamak için, aynı organizatörü kontrol altında aynı vektör olmalıdır. Ilgi TCR insan ise, insan sürekli etki fare sabit etki alanı 9 değiştirilmesi gerekir. Bizim gözlem de tam boy insan TCRS stably fare T-hücrelerinin ifade yoktur. </li><li> Yüksek kalite plazmid DNA Qiagen Maxi Hazırlık Seti veya benzer bir ürün kullanarak hazırlayın. Plazmid konsantrasyonu yaklaşık 1 mcg / ml olmalıdır. </li></ol> 2. Transfeksiyon ve T-hücre İzolasyon Gün -1 (Levha ambalaj hücreleri) <ol><li> Platin-E (Plat-E, Cellbiolabs) tripsin-EDTA ile retroviral ambalaj hücreleri yerinden oynatın ve DMEM medya ile nötralize. </li><li> 5 dakika için 1000 xg&#39;de hücrelerin santrifüjleyin. Süpernatantı aspire ve DMEM medya hücre pelletini tekrar süspansiyon haline getirin. </li><li> Hücre sayısını belirlemek ve 0.6x10 6 / mL hücreleri sulandırmak. Plaka 10mm bir poli-lizin kaplı doku kültürü plakasını hücre süspansiyonu 10 ml ve 37 kuvöz bir hücre gecede büyümek ° C,% 5 CO 2. </li></ol> Gün 0 (Transfeksiyon) <ol start="4"><li> Ertesi sabah, hafif bir mikroskop altında hücrelerin inceleyin. Hücrelerin yaklaşık% 80 konfluent olmalıdır. </li><li> Plaka medya yavaşça çıkarın. 1x PBS ile bir kez yıkayın ve hücrelerin Penisilin-Streptomisin (Pen / Strep) olmadan 10 ml önceden ısıtılmış, taze DMEM medya ekleyin. (Not: Kalem / Strep transfeksiyon engelleyebilir) </li><li> Transfeksiyon kompleksi hazırlayın. A ve B aşağıdaki gibi karışımı hazırlayın: <table border="0"><tbody><tr><td> Plazmid DNA: A karıştırın </td><td> 9 mikrogram </td></tr><tr><td> plazmid PCL-Eco yardımcı </td><td> 6.3 mikrogram </td></tr><tr><td> OptiMEM </td><td> 1,5 mL </td></tr><tr><td colspan="2"></td></tr><tr><td> Mix B: Lipopfectime 2000 </td><td> 60 mcL </td></tr><tr><td> OptiMEM </td><td> 1,5 mL </td></tr></tbody></table> Oda sıcaklığında 5 dakika için ayrı ayrı A ve B inkübe Nazikçe A ve B karıştırın ve transfeksiyon kompleks oluşturacak şekilde oda sıcaklığında 20 dakika inkübe edin. </li><li> Plat-E hücrelerin içine yavaş yavaş karışımı (toplam 3 ml) damla. Yavaşça plaka transfeksiyon karışımı eşit olarak dağıtmak için ileri geri sallayın. </li><li> 37 ° hücreleri inkübe ° C /% 5 CO 2 hücre inkübatör. </li><li> 6-8 saat sonra taze DMEM orta viral üretimi için 10 ml (Kalem / Strep ile) orta yerine. </li></ol> Antikorlar ile Coat plaka <ol start="10"><li> Fare T-hücrelerinin viral transdüksiyon önce aktif hale getirilmesi gerekir. T-hücrelerinin aktive etmek için çeşitli yolları vardır. Burada plaka bağlı anti-CD3e ve anti-CD28 antikorları kullanın. 1 mcg / ml ve anti-CD3e 2 mcg / ml steril PBS anti-CD28 antikor karışımı hazırlayın. </li><li> 24-iyi bir doku kültürü plakasının her bir kuyunun 250 mcL antikor karışımı koyun. Plaka 37 4 ° C veya 2 saat gece kaplı ° C. </li></ol> Fare dalak tek hücre süspansiyonu hazırlanması <ol start="12"><li> Hasat fare dalak ve RPMI medyum transfer. </li><li> Dalak bir hücre süzgeç koyun. 5 cm doku kültürü plaka içine bir şırınga pistonu ezerek dalak. Steril PBS ile hücre süzgeç kapalı hücre durulayın. </li><li> 1000 xg, 5 dakika santrifüjleyin. </li><li> Süpernatant atın. ACK parçalama tampon yeniden süspanse hücre pelet (dalak başına 2 ml). Oda sıcaklığında 2 ila 3 dakika inkübe edin. 20 ml RPMI orta ekleyin ve 5 dakika boyunca 1000 xg santrifüj. </li><li> Süpernatant atın. Steril PBS içinde yeniden süspanse hücre pelet. 4 5 dakika hücre sayısı ve santrifüj ile 1000 xg ° C belirleyin T-hücre izolasyonu devam edin. </li></ol> Fare T-hücrelerinin izolasyonu ve aktivasyon <ol start="17"><li> Manyetik ürün kılavuzuna (CD8 + Miltenyi Biotec T-hücre izolasyon kiti) göre hücrelerin etiket. </li><li> M bir LS sütun (Miltenyi Biotec) yerleştirin.agnetic alanında. Etiketli hücre süspansiyonu sütun üzerine uygulayın. (Fare CD8 + T-hücrelerinin zenginleştirilmiş) ile akış toplayın. Ürün kılavuzuna göre 3 mL tampon sütun üç kez yıkayın ve flow-through önceki birleştirmek. </li><li> Leke ve anti-CD3e ve anti-CD8a antikorlar ile hücreler flow sitometri ile analiz (Şekil 2a). Tipik bir ayırma için, yaklaşık% 8-10 toplam hücreleri izole edilebilir. ~ Izole hücrelerinin% 90, CD8 + T-hücrelerinin. </li><li> 1000 xg, 5 dakika hücreleri santrifüjleyin. Süpernatantı atın. Rekombinant insan IL-2 (rhIL2, 20 ng / ml) 1x10 6 / ml RPMI medyum içinde hücre pelletini tekrar. </li><li> Sadece hücreleri eklemeden önce, plaka (2.11 önceden hazırlanmış) antikor çözüm kaldırmak. Her steril PBS ve kaldır PBS ile iyice durulayın. </li><li> 1 ml hücre süspansiyonu, her bir kuyunun ekleyin. 37 ° C /% 5 CO 2 inkübatör bir hücre tabak koyun. </li></ol> 3. Aktif (Gün 2 Gün 3) Enfeksiyon T-hücrelerinin <ol><li> 2 gün sonra virüs üretim, Plat-E hücre plakasını orta sarı haline gelmelidir. 15 ml konik tüp viral süpernatant aktarın. Daha fazla viral üretim plaka (isteğe bağlı) 10 ml taze DMEM orta ekleyin. </li><li> Hücrelerin enkaz kaldırmak için 5 dakika için 1000 xg&#39;de virüs süpernatant santrifüjleyin. Yeni bir tüp virüs süpernatant dikkatlice aktarın. Borunun alt kısmında bir miktar sıvı bırakın ve hücrelerin enkaz rahatsız etmeyin. </li><li> Toplayın plakadan 50 ml konik tüp içine T-hücrelerinin aktive. Hücre sayısını belirler. (Un transduced) negatif kontrol olarak kullanılmak üzere ayrı bir tüpe bazı hücreler kaydedin. 5 dakika için 1000 xg&#39;de santrifüjleyin ve supernatant atın. </li><li> 20 ng / ml rhIL2 ve 10 mg / ml protamin sülfat ile 1 ml başına 10 6 hücre virüs süpernatant hücre pelletini tekrar. 24 plaka her bir kuyu için 1 ml hücre süspansiyonu ekleyin. Aynı miktarda rhIL2 ve protamin sülfat ile aynı hücre yoğunluğu RPMI orta kontrol tüpü hücrelerin tekrar Hücreler aynı plaka ekleyin. </li><li> Plaka plastik film ile sarın. Herhangi bir fren ile 32 ° C&#39;de 90 dakika, 2000 xg&#39;de santrifüjleyin. </li><li> Santrifüj sonra, plastik folyoyu çıkarın. 1 ml taze RPMI medyum her bir kuyu için 20 ng / ml rhIL2 ekleyin. Inkübatör geri plaka koyun. </li><li> (Isteğe bağlı) TCR Yükseköğretim ekspresyon seviyesi virüs süpernatant toplama ve 3. gününde enfeksiyon prosedürü tekrar elde edilebilir. </li></ol> 4. Hücre Genişleme ve İletimi Verimliliği Değerlendirme <ol><li> Inceleyin günlük T-hücrelerinin transduced. Split gerektiğinde RPMI orta rhIL2 01:03 oranları hücreler. Hücreleri büyümek (orta sarıya döner) izin vermeyin. </li><li> Günde 6 ya da 7 gün, antikor ve T-hücre yüzeyinde TCR ifade değerlendirmek için TCR için özel bir MHC tetramer ile hücre leke. Kullanarak kontrol olarak T hücreleri (Şekil 2c) un transduced. </li><li> T-hücrelerinin transduced daha aşağı uygulamalar için hazır. </li></ol> 5. Temsilcisi Sonuçlar Splenositlerin arıtma olmadan transduced olmasına rağmen transdüksiyon önce T-hücre alt arındırmak için genellikle avantajlıdır. Yüksek saflıkta T-hücre alt grupları, ticari manyetik boncuk (Şekil 2a) kullanılarak elde edilebilir. T-hücrelerinin TCRS ifade düzeyini değerlendirmek için, TCR alfa veya beta zincirleri için özel antikorlar kullanılabilir. Genellikle hücrelerin% 30 -% 80 ifade TCR genleri ve virüs titreleri bağlı olarak TCR transduced (Şekil 2b). MHC tetramer özgü TCR için daha da işlevsel TCRS ifade (Şekil 2c) doğrulamak için kullanılan olabilir. <img src="/files/ftp_upload/2307/2307fig1.jpg" alt="Şekil 1" /> Şekil 1. Örnek T-hücre reseptör geni inşa alt klonlanmış retroviral bir vektör içine kendi kendine cleavable 2A peptid ilintileyici 8 Tam boy alfa ve beta zincirinin genleri ile bağlantılı. <img src="/files/ftp_upload/2307/2307fig2.jpg" alt="Şekil 2" /> Şekil 2. Fare T-hücrelerinin başarılı bir şekilde izolasyonu ve transdüksiyon Örnek (a) CD8 T hücreleri kullanarak splenositlerin CD8a + T Hücre İzolasyon Kiti (Miltenyi Biotec) ve anti-CD3e ve anti-CD8a antikorlar ile boyanarak izole edilmiştir. 209 -217 4 peptid anti-insan Vbeta 8 antikor (b) ve HLA-A2kb gp100 tetramer (c) ve vitray: CD8 T hücreleri izole insan gp100 R6C12 TCR özel transduced edildi.

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	<li>Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+T+cells+'see'+antigen.">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li><li>Berg, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li><li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li><li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li><li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li><li>Szymczak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li><li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin.">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li><li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li></ol>

En iyi sonuçları elde etmek için birkaç adım kritik öneme sahiptir. Plat-E ambalaj hücre hattı sağlıklı bir durumda tutulmalıdır. Gerekirse, hücrelerin retroviral yapısı protein ekspresyonu kaybını önlemek için 1 mcg / ml puromycin, 10 mcg / ml blasticidin DMEM ortamda bir kaç kez geçişli olabilir. Transfeksiyon gününde, hücrelerin ~ 80% birleşmiş olmalıdır. Çok az ya da çok fazla hücreleri virüs titresi azaltabilir. Virüs kalitesi kolayca transduced hücre hatları üzerinde test ederek kontrol edilebilir. TCRS CD3 kompleksleri hücre yüzeyinde ifade edilmesi gerektiğinden, rutin kullanımı 58α-/β- hibridoma hücrelerin 10 (endojen TCR α veya β zinciri ifade, ancak ifade CD3 kompleksi değil bir hücre hattı). Hücreleri virüs süpernatant 1 ml ile uyum hibridoma hücreleri yaklaşık 100% transdüksiyon etkinliğini edinin. Son olarak, T-hücrelerinin aktive retrovirüs sadece aktif hücreler bölünerek bulaşabilir çünkü prolifere gerekir. Bu yöntem, antijen-spesifik T-hücrelerinin bazı sınırlamaları vardır üretmek için buraya sundu. İlk olarak, birincil fare T-hücrelerinin% 100 transdüksiyon etkinliğini ulaşmak için zordur. Hücrelerin bir kısmı faiz TCR ​​ifade yoktur. İkincisi, TCR transduced α ve β zincirleri ilgi TCR ifade düzeyini azaltabilir, endojen TCRS 9 mispair. Buna ek olarak, in vivo 11 mispaired TCRS otoimmünite neden olabilir. Son olarak, sadece sınırlı bir zaman diliminde, T-hücrelerinin kullanılabilir transduced vardır. Yaklaşık bir hafta sonra, hücreler yeniden-uyarılmış sürece apoptoz uğrarlar. Ancak, hücreler tükenebilir ve tekrarlayan yeniden tesir ile fonksiyonları kaybetmek olabilir.

Medya: DMEM orta (DMEM 10% ısı inaktive FBS + Pen / Strep + L-Glu + Sodyum Piruvat + Non-esansiyel amino asit). Pen / Strep olmadan DMEM orta (DMEM orta olarak aynı, ancak Pen / Strep eklemeden). RPMI orta (RPMI 10% ısı inaktive FBS + L-Glu + Non-esansiyel amino asit + β-Mercaphoethanol + Kalem / Strep + Sodyum Piruvat).

retroviral transduction of t-cell receptors in mouse t-cells

T-hücre reseptörleri (TCRS), bağışıklık sisteminde merkezi bir rol oynar. T-hücre yüzeyleri TCRS özellikle peptid antijenleri, antijen sunan hücreler (APC) 1 tarafından sunulan tanıyabilirsiniz . Bu tanıma, T-hücrelerinin (örneğin sitokin üretim, hedef hücrelerinin öldürülmesi) fonksiyonel sonuçlarının bir dizi aktivasyonu yol açar. TCRS işlevsel rolünü anlamak immünoloji ile ilgili hastalıklar (örneğin, kanser veya otoimmünite) çeşitli tedavi etmek için bağışıklık sisteminin gücünü kritik öneme sahiptir. Bu fare modelleri, çeşitli şekillerde yapılabilir TCRS, çalışma için uygundur. TCR transgenik fare modelleri, masraflı ve zaman alıcı ve şu anda 2-4 bunlardan sadece sınırlı sayıda vardır . Alternatif olarak, fareler ile T-hücrelerinin antijen spesifik chimera kemik iliği tarafından oluşturulan olabilir. Bu yöntem aynı zamanda birkaç hafta sürer ve uzmanlığı 5 gerektirir . , In vitro aktive fare T-hücrelerine TCRS retroviral transdüksiyon istenen peptid-MHC özgüllüğü T hücreleri elde etmek için hızlı ve nispeten kolay bir yöntemdir. Antijen spesifik T-hücrelerinin, bir hafta içinde oluşturulan ve herhangi bir alt uygulamalarda kullanılabilir. T-hücrelerinin transduced incelenmesi insan immünoterapi doğrudan uygulama, insan T-hücrelerinin evlatlık transferi olarak antijen-spesifik TCRS transduced, kanser tedavisi 6 için gelişmekte olan bir stratejidir . Burada retrovirally, in vitro aktive fare T-hücrelerinin içine TCRS uyum sağlamak bir protokol mevcut . Insan ve fare TCR genleri her ikisi de kullanılabilir. Spesifik TCR genleri taşıyan Retrovirüsler üretilen ve fare anti-CD3 ve anti-CD28 antikorları ile aktive T hücreleri enfekte etmek için kullanılır. T-hücrelerinin, in vitro genişleme transduced sonra flow sitometri ile analiz edilir.

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

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

Biz retroviral transdüksiyon kullanarak fare antijen spesifik T-hücrelerinin üretmek için bir protokol mevcut

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 ...

retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

Research

JoVE Journal

Immunology and Infection

34.8K Görüntüleme.  New York University School of Medicine. Biz retroviral transdüksiyon kullanarak fare antijen spesifik T-hücrelerinin üretmek için bir protokol mevcut

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

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&#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 ...

İmmünoloji ve Enfeksiyon

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 ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Naif CD4 adenoviral İletimi Treg Farklılaşma Eğitim Hücreler T

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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 ...

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&#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 ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Kemik iliği projenitör hücrelerinin retroviral İletimi T-hücre reseptör Retrogenic fareler üretmek için

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Yaratma<em&gt; De Novo</em&gt; Merkezli Hemichain retroviral transdüksiyonu ile antijen-özel insan T hücre reseptörleri

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Eğitim için bir genetik Yaklaşım olarak yardımcı T hücrelerinin retroviral İletimi Mekanizmaları kendi Farklılaşma ve Kontrol Fonksiyonunun

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Bağırsak-Homing Düzenleyici T Hücre İndüksiyon In Vivo Augmentation

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Akış Sitometri Analizi için Fare Böbrek-Resident CD8+ T hücrelerinin izolasyonu

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...