Biz Aaron Selya yararlı tartışma için teşekkür etmek istiyorum. Bu çalışma, JS, MO bir pilot hibe ve Johns Hopkins Sıtma Araştırma Enstitüsü, Savunma hibe PC 040.972 Departmanı, Ulusal Sağlık Enstitüsü, hibe AI29575, CA108835, AI077097 tarafından desteklenen

1. , HLA-A2-Ig tabanlı aAPC <ol><li> Steril borat tamponu hazırlayın <ol><li> Borik asit, 0.1M yapmak için suda çözülür. PH 7.0 &#39;ye ayarlayın. </li><li> Filtre 0.22μm steril bir filtre ve mağaza üzerinden 4 ° C </li></ol></li><li> Steril Boncuk Yıkama tamponu hazırlayın <ol><li> 956ml PBS w / o magnezyum veya kalsiyum alın. </li><li> 30ml insan AB serum ekleyin. </li><li> EDTA 2mm son konsantrasyon ekleyin. </li><li> 0.1gsodium azid ekleyin. </li><li> 0.22 mikron steril filtre ve mağaza süzülür 4 ° C </li></ol></li><li> , Stok, yaklaşık 400 milyon boncuk (boncuk hemasitometre sayılabilir) 1ml Invitrogen M-450 Epoksi boncuk alın, ve steril, vida top cam flakon koymak. </li><li> Boncuklar flakonun tarafına bağlı ise, bir Dynal mıknatıs MPC-1 karşı flakon koyun, aspirasyon ile süpernatantı çıkarın. Boncuk, borat tamponu 1ml ile bir kez yıkayın. </li><li> 1 ml borat tamponu ve HLA-A2-Ig dimer ve anti-insan CD28 mAb (Klon 9.3) 20 mg 20 mg bir karışımı süspanse edin boncuk. </li><li> Boncuk konjugasyon Protein: Rotator Cam şişe koymak ve 4 döndürmek ° C 24 saat. </li><li> MPC-1 mıknatıs tüp yerleştirin ve tüm borat tamponu çıkarın. </li><li> Boncuklar iki kez 1ml Boncuk Yıkama tamponu ile yıkayın. </li><li> 1ml Boncuk Yıkama tamponu boncuk inkübe, 4 döndürmek ° C 24 saat boyunca. Boncuk Yıkama tampon insan AB serum içerdiğinden, boncuklar üzerinde kalan protein bağlanma yeri engeller. </li><li> Cam şişeden süpernatantı 1ml taze Boncuk Yıkama tamponu ile değiştirin. </li></ol> 2. Kalite kontrol aAPC ve peptid yükleme, depolama <ol><li> FACS tüpler tampon yıkama 100μl FACS ~ 5 × 10 5 boncuk ekleyin ve anti-fare IgG1-PE 1μl (HLA-A2-Ig Fc bölümünü tanımaz) ve anti-fare IgG2a-FITC 1μl (tanımak leke anti-CD28 mAb Fc parçası). 20 dakika boyunca tampon yıkama FACS boyama sonra, akış sitometresinin (Şekil 4) hemen boyama sonucu tekrar yıkayın ve okuyun. </li><li> Boncuklar üzerine Peptid yükleme: 1 ml steril PBS ile cam flakon boncuklar iki kez yıkayın. 1ml steril PBS ile süspanse edin sonra CMV peptid (1mg/ml) 10μl eklemek </li><li> Hemasitometre boncuk sayın ve tarih ve konsantrasyon ile, tüp etiketi. </li><li> aAPC HLA-A2-Ig dimer üzerine peptid bağlama yeterli zaman tanımak için en az 3 gün, 4 peptid inkübasyon ° C, sonra kullanıma hazır. Boncuk 4 saklanabilir ° C ve en az 6 ay süreyle işlevsel kalır. </li></ol> 3. İnsan CTL izolasyon <ol><li> 10 BD Sodyum Heparin Vacutainer tüpler içine sağlıklı HLA-A2 pozitif vericiden ~ 100 ml taze periferik kan toplayın. 21-gauge iğne veya hemoliz önlemek için büyük kullanın. </li><li> 10 dakika boyunca oda sıcaklığında 300xg santrifüjleyin </li><li> Aspirasyon üst plazma tabakasını dikkatlice çıkarın. Plazma kültür ortamı için ek olarak kullanılabilir. </li><li> Steril PBS ile toplanan plazma değiştirin ve steril bir T75 kültür şişelerinde veya 50ml konik tüpler içine kan transferi. Yukarı ve aşağı pipetleme kan PBS ile karıştırın. </li><li> Bir kez tüm kan toplanır, dört ek 50ml konik tüpler hazırlamak ve Ficoll-Paque Plus 15ml ekleyin. </li><li> Yavaş yavaş bindirme Ficoll her tüpün üstüne kan hücreleri 30-35ml. Ficoll ve kan hücreleri arasında arayüz ayrı tutun. </li><li> Oda sıcaklığında 20 dakika 500xg santrifüjleyin. Fren &quot;kapalı&quot; ve katmanları arasında açık bir arayüz sağlamak için mümkün olduğunca düşük ivme açın. </li><li> Seriological pipet kullanarak, dikkatle PBMC tabakası aspire taze 50ml konik tüp içine PBMC toplamak. Tüm PBMC 400xg az 10 dakika süreyle PBS ve spin 30ml eklemek hasat edilir. Süpernatantı atın ve kalan tüm Ficoll kaldırmak için bir kez daha 30ml PBS ile yıkayın. </li><li> Miltenyi insan CD8 + T hücre üretici firmanın protokolüne göre izolasyon kiti kullanılarak CD8 + T hücre izolasyonu geçin. Hücreleri - Bu kit çok CD8 tüketen (genellikle&gt;% 95) CD8 + hücreler için zenginleştirir. </li><li> CD8 + T hücreleri sayın. Beklenen saflığı% 95 daha büyük olmalıdır. Onaylamak ve bu kullanım 2x10 5 hücre CD4/3/8 FACS analizi gerçekleştirmek için. Ya kalan CTL antijen-spesifik aAPC uyarılması için hemen olabilir ya da gelecekte kullanılmak üzere dondurularak muhafaza edilebilir. </li></ol> 4 in vitro aAPC temelli bir kültür sistemi <ol><li> Kültür ortamı hazırlayın <ol><li> TF (laboratuar 4 T hücre büyüme faktörü) 2X kültür ortamı: tam RPMI orta artı% 5 plazma ve% 8 T hücre büyüme faktörü otolog donör. </li><li&gt; Donör otolog plazma AB serumu ısı ile inaktive edilmiş insan tarafından ikame edilebilir. </li></ol></li><li> Tam RPMI medyum TF 2X kültür ortamı artı 8 ml 8 ml yeniden süspanse 1 milyon CTL, 1 × 10 6 aAPC, iyice karıştırın. </li><li> Iyi 96 U altındaki doku kültürü plakaları üzerine plaka hücrelere bir multi-kanal pipetter kullanın. (Için de ul 160) </li><li> Kültür hücreleri 37 ° C, 7 gün süreyle% 5 CO 2 incubater. 4 gün 80 ul / TF 2X orta hücrelerin besleyin. </li><li> Hücreler 7 gün hasat edilebilir hazırız. Hasattan sonra, mıknatıs karşı hücreleri yer ve eski aAPC çıkarın. </li><li> Antijen özgüllüğü, üretici firmanın protokolüne göre tetramer boyama ile tespit edilebilir. Fenotip boyama ve hücre içi sitokin boyama önceki çalışmada 3 göre yapılmaktadır. </li><li> Hasat hücreleri aynı koşullar altında tekrar aAPC replated olabilir. Hücre sayısı ve antijen özgüllüğü tekrarlanan stimülasyon sonrasında artması beklenmektedir. </li></ol> 5. Temsilcisi Sonuçlar: HLA-A2-Ig ve anti-CD28 konjugasyon sonra aAPC bir örnek Şekil 4&#39;te gösterilmiştir. Başarılı protein konjugasyon net bir kayma ile ilgili antikor boyama açıktır. Periferik kan CMV spesifik CTL frekans aAPC aracılı stimülasyon tek bir hafta sonra, genellikle% 0.5-1 iken, özgüllüğü 55 ulaşabilirsiniz -% 93 (Şekil 2 ve 3). Antijen spesifik CTL genişleme farklı bağışçılar arasında çok değişken olabilir, ancak sonuçlar aynı donör içinde tekrarlanabilir. Ekstrapolasyon, CMV hücrelerin genişlemesi habercisi seviyeleri doğrudan ex vivo (veriler gösterilmemiştir) ile karşılaştırıldığında binlerce kat olabilir. Hücre içi sitokin boyama (Şekil 3) Bu genişletilmiş CTL polifonksiyonel ziyade, bitkin, uzun süreli hücre kültürü ve önemli çoğalması. <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="Şekil 1"/> Şekil 1 allojeneik HKHT evlatlık immünoterapi için insan CTL aAPC ex vivo genişlemesi Temsilcisi akış şeması <img src="/files/ftp_upload/2801/2801fig2.jpg" alt="Şekil 2"/> Şekil 2 CMV spesifik CTL bir hafta sonra kültür aAPC tarafından oluşturulan Temsilcisi tetramer boyama sonucu <img src="/files/ftp_upload/2801/2801fig3.jpg" alt="Şekil 3"/> Şekil 3 CMV spesifik CTL aAPC tarafından üretilen Temsilcisi hücre içi sitokin boyama sonuç (CMV özgüllük% 61) <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="Şekil 4"/> Şekil 4. M-450 Epoksi boncuk Temsilcisi boyama sonucu protein konjugasyon sonra anti-fare IgG1-PE ve anti-fare IgG2-FITC ile boyandı

<ol>
	<li>Walter, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N Engl J Med. 333, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

Biz burada açıklamak aAPC sistemi, çeşitli antijenlere karşı insan CTL ex vivo genişlemesi için verimli bir sistem. Özel bakım 96-plaka kültürü, kalitesi ve protein konjugasyon aAPC ve CTL, hatta dağıtım konusunda alınmalıdır. Biz bir milyon kat 4 antijen-spesifik CTL kadar genişletilebilir sırasında fazla 8 hafta boyunca CTL genişletmek mümkün olmuştur bu yaklaşımı kullanarak. Hücre hatları veya diğer acellular platformlarda 5 yararlanarak çeşitli yapay APC sistemleri var olmuştur, ancak her sistem, genişleme ve özgüllüğü konusunda farklı uygulamaları destekleyen ile eşsiz bir profile sahiptir yayınlanan veriler göre . KVK, kalite, miktar olarak önemli olduğundan da önemlisi, sistem tarafından üretilen CMV spesifik CTL polyfunctionality üstün anti-viral etkinliği tanımak bekleniyor.

<table border="1"><tbody><tr><th> Reaktif </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Vacutainer tüp (heparin içeren) </td><td> Becton Dickinson </td><td> 367874 </td></tr><tr><td> İnsan CD8 + T hücre izolasyon kiti </td><td> Miltenyi </td><td> 130-094-156 </td></tr><tr><td> Dynabeads M-450 Epoksi </td><td> Invitrogen </td><td> 140,11 </td></tr><tr><td> Dynal MPC-1 Mıknatıs </td><td> Invitrogen </td><td> 120-01d </td></tr><tr><td> Ficoll-Paque Plus </td><td> GE Healthcare </td><td> 17-1440-03 </td></tr><tr><td> RPMI medyum 1640 </td><td> Gibco </td><td> 11875 </td></tr><tr><td> HLA-A2-Ig dimer X </td><td> Becton Dickinson </td><td> 551263 </td></tr><tr><td> iTAgMHC tetramer (HLA-A2-CMV)-PE </td><td> Beckman Coulter </td><td> T20099 </td></tr><tr><td> Falcon net bir 96-iyi Microtest plaka </td><td> Becton Dickinson </td><td> 353077 </td></tr><tr><td> Rat anti-fare IgG2a-FITC </td><td> Becton Dickinson </td><td> 553390 </td></tr><tr><td> Keçi anti-fare IgG1-PE </td><td> Invitrogen </td><td> P21129 </td></tr><tr><td> İnsan serum AB tipi </td><td> Atlanta biyolojik </td><td> S40110 </td></tr><tr><td> Fare, anti-insan CD8a-FITC </td><td> Sigma-Aldrich </td><td> F0772 </td></tr><tr><td> Fare anti-insan CD8a APC </td><td> Becton Dickinson </td><td> 340684 </td></tr><tr><td> Fare anti-insan IFNγ-FITC </td><td> Becton Dickinson </td><td> 340449 </td></tr><tr><td> Fare insan anti-TNF-PE </td><td> Becton Dickinson </td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

En iyi effektör fonksiyonu ile CTL, çeşitli hücre içi enfeksiyonlara ve kansere karşı koruma aracılık kritik rol oynar. Ancak, bireylerin baskılayıcı bağışıklık mikroçevresinin gösterebilir ve aktive CTL aksine, otolog antijen sunan hücreler tolerize eğilimi olabilir veya antijen spesifik CTL anergize. Sonuç olarak, hala deneysel aşamada olmasına rağmen CTL tabanlı evlatlık immünoterapi gibi kanser ve virüs enfeksiyonları gibi çeşitli hastalıklar için umut verici bir tedavi haline gelmifltir. İlk deneylerde ex vivo genişletilmiş CMV (sitomegalovirüs) immün sistemi baskılanmış allojenik kemik iliği nakli hastalarında CMV enfeksiyonu tedavisi için kullanılan spesifik CTL edilmiştir. Bu hastalarda yaşamı tehdit eden CMV viremi ortak, alan hastaların hiçbiri CTL anti-CMV bağışıklık adoptively transfer CTL 1 tarafından kurulmuştur ima, CMV ile ilgili hastalık geliştirmek genişletti. Melanom için umut verici sonuçlar da gözlenmiştir ve diğer kanser türleri 2 için uzatılabilir. Ex vivo insan CTL teşvik etmek ve genişletmek için birçok yol olmasına karşın, güncel yaklaşımların maliyet ve teknik sınırlamalar ile sınırlıdır . Örneğin, mevcut altın standarttır otolog DC dayanmaktadır. Bu, her bir hastanın önemli bir lökosit sayısı bağış gerektirir ve aynı zamanda çok pahalı ve zahmetli bir iştir. Ayrıca, bu sadece suboptimal effektör fonksiyonu 3 DC genişletilmiş CTL in vitro karakterizasyonu ayrıntılı sahip olduğunu ortaya koymuştur. Burada, evlatlık immünoterapi (Şekil 1) insan CMV spesifik CTL ex vivo genişlemesi için son derece verimli aAPC tabanlı bir sistem sunuyoruz. AAPC insan HLA-A2-Ig dimer ve anti-CD28mAb 4 hücre ölçekli manyetik boncuk kaplin tarafından yapılmıştır. AAPC yapıldıktan sonra, çeşitli ilgi peptitler ile yüklü ve ay için işlevsel kalır olabilir. Bu raporda, aAPC CMV, pp65 (NLVPMVATV) baskın bir peptit ile yüklendi. AAPC ile bir hafta boyunca sağlıklı bir donörden insan CD8 + CTL saflaştırılmış kültür sonra, CMV spesifik CTL, özgüllüğü% 98 (Şekil 2), dramatik bir şekilde artmış ve 10.000 &#39;den fazla kat güçlendirilmiş olabilir . CMV spesifik CTL gerekiyorsa, daha fazla genişleme aAPC ile tekrarlayan stimülasyonu ile kolayca elde edilebilir. Fenotipik ve fonksiyonel karakterizasyonu bu genişletilmiş hücreleri efektör bellek fenotipi (Şekil 3) TNF ve IFNγ hem de önemli miktarda ve gösterir.

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antijen-spesifik T hücrelerinin indüksiyon ve genişleme için yeni bir DC bağımsız bir yöntem açıklanmıştır. HLA-A2-Ig tabanlı yapay Antijen sunan hücreler (aAPC), HLA-A2 verimli farklı antijen özgüllüğü CTL genişletmek için kısıtlı peptitler ile yüklenir. Bu teknoloji tabanlı CTL evlatlık immünoterapi için büyük bir potansiyele sahiptir.

Verimli HLA-Ig Tabanlı Yapay antijen sunan hücrelerin Ex vivo</emİnsan CTL> Genleşme

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

hla-ig-based-artificial-antigen-presenting-cells-for-efficient-ex

Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

15.3K Görüntüleme.  Johns Hopkins University. Antijen-spesifik T hücrelerinin indüksiyon ve genişleme için yeni bir DC bağımsız bir yöntem açıklanmıştır. HLA-A2-Ig tabanlı yapay Antijen sunan hücreler (aAPC), HLA-A2 verimli farklı antijen özgüllüğü CTL genişletmek için kısıtlı peptitler ile yüklenir. Bu teknoloji tabanlı CTL evlatlık immünoterapi için büyük bir potansiyele sahiptir.

Video: HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antijen Özgül In Vivo</emKAKE Etiketli Hedef Hücreler> Killing Testi

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

İmmünoloji ve Enfeksiyon

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Bryostatin 1/Ionomycin Yoluyla Tümör-reaktif T hücrelerinin ex vivo Genişleme ve Ortak Gama Zinciri Sitokinler Formülasyon

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

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 ...

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 ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Yapay Antijen Hücre (AAPC) Aracılı Aktivasyon ve Doğal Öldürücü T hücreleri genişletilmesi Presenting

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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 ...

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

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

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

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

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

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

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Etkin Killer yapay antijen sunan hücreler (KaAPC)<em&gt; İn Vitro</em&gt; İnsan Antijen-spesifik T hücrelerinin tükenmesi

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Fenotipik Analizi ve İnsan Cilt lenfosit izolasyonu<em&gt; Ex Vivo</em&gt; Hücre Kültürü

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Bir Antijen odaklı Kolit Modelinin Geliştirilmesi Antijen T hücreleri için Hücreler sunulması ile Antijen Sunumu Eğitim için

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Verimli HLA-Ig Tabanlı Yapay antijen sunan hücrelerin Ex vivo Genleşme