Yazarlar Poly-ICLC armağanı için Andres Salazar (Oncovir, Inc) teşekkür etmek istiyorum.

1. PBMC 4 İzolasyon ve Kriyoprezervasyon <ol><li> Aseptik başak bir plazma transfer seti kullanarak lökaferezis torbaya erişim limanlarından biri. 60 ml&#39;lik şırınga kullanarak, steril bir 500 ml&#39;lik şişe içine hastadan elde edilen lökaferezis aktarın. </li><li> Oda sıcaklığında RPMI kullanarak orijinal ses 2x lökaferezis hacmi ayarlayın. Iyice karıştırın. </li><li> Yavaşça Ficoll-Paque PLUS şişe karıştırın. Steril bir 50 ml&#39;lik konik bir tüp içine 12 ml Ficoll Paque Plus eklenmesi. </li><li> Ficoll içeren her bir steril 50 ml konik tüp seyreltilmiş lökaferezis ürünün yavaşça tabakası 30 mi. Ficoll ve hücre süspansiyonu arasındaki arayüz rahatsız etmemek için dikkatli olun. </li><li> Lökaferezis tüm katmanlı edilene kadar adımları 1.3 ve 1.4 tekrarlayın. </li><li> , Fren ile oda sıcaklığında 20 dakika boyunca 1000 x g &#39;de 50 ml konik tüp santrifüjleyin. </li><li> Dikkatle her tüp ve transfe gelen PBMC&#39;lerin bulutlu tabakası hasatyeni bir steril 50 ml tüp r. </li><li> 50 ml lik bir son hacme kadar her bir tüpe RPMI ekleyin. Tersine çevirerek yavaşça karıştırın. </li><li> 4 10 dakika ° C tam fren ile 500 × g hücreleri santrifüj. </li><li> Her bir tüpten süpernatantlar çıkarın. Her bir tüp, hücre topakları yeniden süspanse edin ve 50 ml &#39;lik bir son hacim RPMI ekleyin. Tersine çevirerek yavaşça karıştırın. </li><li> 4, 6 dakika boyunca 500 ° C x g&#39;de santrifüjleyin hücrelerin </li><li> Her bir tüpten süpernatantlar çıkarın. Süspanse edin ve bir adet 50 ml&#39;lik konik bir tüp içine birlikte yüzme hücre süspansiyonları. </li><li> Fazla yer RPMI ile 50 ml toplanmış PBMC hacmi getirin. Tersine çevirerek yavaşça karıştırın. </li><li> 4, 6 dakika boyunca 300 ° C x g&#39;de santrifüjleyin hücrelerin </li><li> Süpernatantı. 50 ml RPMI hücre pelletini. Tek tip hücre süspansiyonu sağlamak için hafifçe karıştırın. </li><li> Hücrelerin sayısını ve hücre canlılığı belirler. Canlı hücrelerin toplam sayısını hesaplayın. </li><li> 300 hücreleri santrifüj4, 6 dakika boyunca x g ° C Dondurma ortamı hazırlayın. Insan AB serumu (Valley Biyomedikal) içinde, dondurma ortamı% 10 dimetil sülfoksit (DMSO Miltenyi Biotec) oluşur. </li><li> Süpernatantı. 2 x 10 8 hücre / soğuk dondurma ortamı içinde ml &#39;lik bir nihai konsantrasyonda tekrar süspansiyon hücreleri. </li><li> 1.8 mi cryovials 1 ml&#39;lik olun. </li><li> Kontrollü oranı dondurucu içine cryovials aktarın ve donma koşmak, Program 1 başlar. Miktarı sonunda, hemen sıvı nitrojen dondurucunun buhar fazına dondurulmuş kriyotüplerin aktarın. </li></ol> 2. Gün 0: monositler gelen dendritik hücreler farklılaşması <ol><li> , Steril bir 50 ml konik tüp, 30 ml RPMI /% 1 otolog plazma ekleyin. </li><li> 37 ° C su banyosunda yavaşça sallanarak dondurulmuş PBMC&#39;ler çözülme hacimde. Tamamen çözülmüş olduğunda, steril bir 50 ml&#39;lik konik bir tüp için flakon içeriği aktarın. Iyice karıştırın. </li><li> 6 dakika için hücreler santrifüjeoda sıcaklığında, 500 x g &#39;de. Süpernatantı ve RPMI /% 1 otolog plazma küçük bir hacim içinde tekrar süspansiyon pelet hücreleri. Daha sonra 50 ml nihai hacme kadar fazla RPMI /% 1 otolog plazma ekleyin. Iyice karıştırın. </li><li> 6 dakika 500 x g, oda sıcaklığında tekrar hücreleri santrifüjleyin. Süpernatan aspire ve 50 ml RPMI /% 1 otolog plazma pelletini. Iyice karıştırın. </li><li> Hücrelerin sayısını ve hücre canlılığı belirler. Canlı hücrelerin toplam sayısını hesaplayın. </li><li> Levha 40 RPMI 1.40 ml / 225 cm 2 EasyFlask üzerine% 1 otolog plazma x 10 8 hücre. Tüm hücreleri kaplama edilene kadar tekrarlayın. </li><li> 1 için yan düz EasyFlasks inkübe - 37 2 saat ° C doku kültürü inkübatör monositler balona bağlı kalmanıza olanak sağlar% 5 CO 2 içeren içinde. </li><li> Kuluçka tamamlandıktan sonra, inkübatör EasyFlask kaldırmak ve yapışmayan hücreler USI çıkarmak için iki kez yıkayınönceden ısıtılmış RPMI ng 30 ml. </li><li> İkinci yıkamadan sonra, 400-1,000 IU / ml IL-4 ve 100-1.000 IU / ml GM-CSF, 5-8, 40 ml RPMI /% 1 otolog plazma ekleyin. Bu protokol için, 400 IU / ml IL-4 ve 100 IU / ml GM-CSF kullanılarak uygulanır. </li><li> 37 ° C&#39;de 2 gün boyunca inkübe EasyFlasks ° doku kültürü inkübatöründe C,% 5 CO2 içeren. </li></ol> 3. Gün 2: IL-4 ve GM-CSF ile dendritik hücrelerin beslenmesi <ol><li> Her EasyFlask için 400-1,000 IU / ml IL-4 ve 100-1.000 IU / ml GM-CSF ile 4 ml RPMI /% 1 otolog plazma ekleyin. Dönen ve yan sallayarak karıştırın. </li><li> 37 ° C, doku kültürü inkübatörü içinde% 5 CO2 içeren daha fazla 3 gün boyunca EasyFlasks (Gün 5 kadar) inkübe edin. </li></ol> 4. 5. Gün: Olgunlaşmamış dendritik Hücre Hasat <ol><li> Şiddetle şişeler dönen tarafından ve yapışmayan ve gevşek yapışık hücrelere tekrar süspansiyon ve aşağı yukarı pipetleme kültürler hasat. TransferYeni bir 50 ml konik tüplere hücreler hasat. </li><li> Oda sıcaklığında 6 dakika boyunca 500 x g&#39;de santrifüj tüpleri. Süpernatantlar çıkarın ve 50 ml RPMI /% 1 otolog plazma hücre pelletini. Iyice karıştırın. </li><li> Hücrelerin sayısını ve hücre canlılığı belirler. Canlı hücrelerin toplam sayısını hesaplayın. </li><li> Oda sıcaklığında 6 dakika boyunca 500 x g&#39;de santrifüj tüpleri. Plaka 1-2 x 10 400 IU / ml IL-4 ve 6-çukurlu doku kültür plakaları içinde 100 IU / ml GM-CSF ile 3 ml RPMI /% 1 otolog plazma içinde oyuk başına 6 hücre. </li><li> Plakalar 37 ° C&#39;de doku kültürü inkübatörü 2 gece boyunca% 5 CO içeren. </li></ol> 5. 6. Gün: dendritik hücreler olgunlaşması ve Antijen Yükleniyor <ol><li> 10 ug / ml KLH&#39;ye 6 oyuklu kültür plakaları içinde kuyu 1/3 ekleyin. Girdap plakaları karıştırmak için. KLH sadece ilk DC aşı için kullanılır, çünkü sadece KLH kuyu 1/3 eklenir. Sonraki DC aşıları conyalnızca tümör antijen peptidleri tain. </li><li> DC farklı uyaranlara kullanarak olgun teşvik edilebilir. Klinik çalışmalarda kullanılmış olan en yaygın olgunlaşması uyaran bir sitokin kokteyli (TNFa, IL-1 β, ve IL-6) ve prostaglandin E2 (PGE2) 9,10 olmuştur. Daha yakın zamanlarda, Toll-benzeri reseptör (TLR) agonistlerinin kullanımı popülerlik kazanmıştır 11,12. Bu protokol, kuyulara 2 mg / ml Polyinosinic-polisitidilik Asit-poli-L-lisin Karboksimetilselüloz (Poli-ICLC) ekleyin ve iyice karıştırın. Poli-ICLC poli-L-lisin-* ve karboksimetilselüloz (Oncovir, Inc tarafından imal edilmektedir) ile stabilize Poli-IC klinik düzeyde bir formülasyondur. </li><li> Her bir çapraz rekabete 13 önlemek için, yalnızca tek bir peptid alıcı, kendi belirlenmiş oyuklara 100 ug / ml uzun peptid antijenleri (NY-ESO-1 ve / veya Melan-A/MART-1) ekleyin. </li><li> Levhalar 37 ° C&#39;de inkübe edin doku kültürü kuluçka% 5 CO içeren ° C &lt;alt&gt; 2 O / N </li></ol> 6. Gün 7: dendritik hücreler, Kriyoprezervasyon <ol><li> Otolog plazma ve% 10 DMSO kullanarak DC Donma Çözüm hazırlayın. Herhangi bir parçacık halinde malzemeyi çıkarmak için 4 ° C&#39;de 20 dakika boyunca 2,000 DC at dondurma solüsyonuna x g santrifüj edilir. Yeni etiketli 15 ml konik tüp süpernatant aktarın. 4 Mağaza ° kullanana kadar C. </li><li> gecelik inkübasyon tamamlandıktan, hasat ve havuz 50 ml konik tüpler içine peptidler ile darbeli DC içeren tüm kuyuları. </li><li> 6 dakika boyunca 500 x g&#39;de santrifüj tüpleri. Süpernatantlar çıkarın. 5 ml RPMI /% 1 otolog plazma hücre pelletini. Kadar RPMI /% 1 otolog plazma ile 50 ml toplam hacmi getirmek. Iyice karıştırın. </li><li> Hücrelerin sayısını ve hücre canlılığı belirler. Canlı hücrelerin toplam sayısını hesaplayın. </li><li> , Oda sıcaklığında 6 dakika boyunca 500 x g&#39;de santrifüj tüpleri. Süpernatantlar çıkarın ve 5 ml Steri her pelletinile% 0.9 NaCl, USP ve yeni 15 ml konik tüpler transfer. Daha steril% 0.9 NaCl, USP 14 ml hücre süspansiyonu her getirin. Tersine çevirerek kapağı ve karıştırın. </li><li> Önceki adımı iki kez tekrarlayın. Son santrifüj işleminden sonra, pelet bozmadan mümkün olduğunca yakın hücre pelletleri elde etmek, süpernatantlar çıkarın. </li><li> 4 de DC Donma Çözüm hücre pelet tekrar süspansiyon - 20 x 10 6 hücre / her 1.8 ml CryoTube flakon ml ve kısım 1 ml. </li><li> DC aşıların alikotları aşağı dondurma ve uzun süreli depolama için bir sıvı azot dondurucu buhar fazına hemen aktarmak için, kontrollü oranı dondurucu, Program 1 kullanın. </li></ol> 7. Çok Release Test <ol><li> DC aşı her parti değerlendirilir ve genellikle 5 ila 6 hafta aşı hazırlandıktan sonra çalışmaya hasta, içine enjeksiyon için serbest bırakılmadan önce belirli çok bırakma kriterleri (Tablo 1) karşılamak gerekir. </li><li> Canlılık:Otomatik kan sayım cihazının kullanıldığı DC aşı canlılığı değerlendirin. Kabul edilebilir minimum canlılık özellikleri&gt;% 70&#39;tir. </li><li> Kimlik: flow sitometri ile olgun DC&#39;ler (CD11c + CD14-CD83 +) kimliğini değerlendirin. CD11c + hücrelerin yüzdesi,&gt;% 50 olmalıdır. CD11c + ve CD14 + hücrelerin yüzdesi olmalıdır &lt;% 30 ve CD11c&#39;nin yüzdesi +, CD14 ve CD83 + hücre olmalıdır&gt; 50%. </li><li> Sterilite: doğrudan kültür ve Gram boyama ile anaerobik ve aerobik bakteri ve mantar varlığı için test DC aşı. Doğrudan bir kültür sistemi ve endikatör hücre hattı, DNA florokrom boyama Testi kullanarak Mycoplasma varlığı açısından değerlendirir. Bu testler için, asgari özellikleri ayarlamak hiç büyüme tüm kültürlerden görülmektedir olmasıdır. </li><li> Endotoksin: Kinetik-QCL kinetik kromojenik LAL testi kullanılarak endotoksin içeriği değerlendirin ve asgari kriterleri karşılamak için &lt;50 EU / ml olmalıdır. </li><li&gt; Fonksiyon: allojenik T hücreleri ile inkübasyon karışık lenfosit reaksiyon DC fonksiyonu değerlendirin. Uyarılmamış DC&#39;leri ile karşılaştırıldığında proliferasyon varlığı (Şekil 3) rapor edilir. </li></ol>

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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+broad+T-cell+immunity+in+humans+after+a+single+injection+of+mature+dendritic+cells.">Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.</a> J. Clin. Invest. 104, 173-180 (1999).</li><li>Schuler-Thurner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+induction+of+tumor-specific+type+1+T+helper+cells+in+metastatic+melanoma+patients+by+vaccination+with+mature,+cryopreserved,+peptide-loaded+monocyte-derived+dendritic+cells.">Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells.</a> J. Exp. Med. 195, 1279-1288 (2002).</li></ol>

Yazarlar aşağıdaki maddi destek aldık: NIH (AI061684, AI071078, AI044628 ve K08 AI084578 (Miller)), Bill ve Melinda Gates Vakfı, Doris Duke Vakfı, Kanser Araştırma Enstitüsü, Lupus Araştırma İttifak ve Zümrüt Vakfı. Nina Bhardwaj hazırlanması ve bağışıklık işlemek için dendritik hücrelerin kullanımı ile ilgili patentleri bir coinventor olduğunu. Yazarlar herhangi bir diğer ilgili bağlantıları ya da bir finansal faiz veya ayrı ifşa olanlardan el yazması tartışılan konu veya malzeme ile mali çatışma ile herhangi bir kuruluş veya kurum ile finansal katılımı var.

Faz I ve II, monosit türevli DC&#39;lerin klinik çalışmalarda, hastalar, ancak klinik başarıya 1 sınırlı olmuştur bağışıklık tepkileri ortaya göstermiştir. Bu tümör immünoterapötik kullanım için en uygun DC oluşturmak için nasıl fikir birliği eksikliği kısmen olabilir. Klinik dereceli DC oluşturmak için birçok yolu olmasına rağmen, bu yöntemler monositler, olgunlaşma ikna etmek için kullanılan uyarı ve antijen yükleme yöntemleri ayırt etmek için kullanılan sitokinle...

<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Reagent/Supplies/Equipment</td>
 <td>Manufacturer</td>
 <td>Catalog No.</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI-1640 medium with L-glutamine</td>
 <td>BioWhittaker</td>
 <td>12-702F</td>
 <td></td>
 </tr>
 <tr>
 <td>1M HEPES buffered saline</td>
 <td>BioWhittaker</td>
 <td>17-737E</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>BioWhittaker</td>
 <td>17-516F</td>
 <td></td>
 </tr>
 <tr>
 <td>Human albumin, 25% solution USP</td>
 <td>Aventis Behring</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ficoll-Hypaque PREMIUM</td>
 <td>GE Healthcare</td>
 <td>17-5442-03</td>
 <td></td>
 </tr>
 <tr>
 <td>Human AB serum</td>
 <td>Valley Biomedical</td>
 <td>HP1022</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile saline USP</td>
 <td>Hospira</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CryoMACS DMSO </td>
 <td>Miltenyi Biotec</td>
 <td>170-076-303</td>
 <td></td>
 </tr>
 <tr>
 <td>Leukine GM-CSF, 0.5 mg/ml</td>
 <td>Berlex</td>
 <td>A02266</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS GMP IL-4</td>
 <td>Miltenyi Biotec</td>
 <td>170-076-101</td>
 <td></td>
 </tr>
 <tr>
 <td>Hiltonol, Poly-ICLC, 2 mg/ml</td>
 <td>Oncovir</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>VACMUNE KLH</td>
 <td>Biosyn</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>225 sq cm EasyFlasks</td>
 <td>Nalgene Nunc</td>
 <td>159934</td>
 <td></td>
 </tr>
 <tr>
 <td>Falcon 6-well tissue culture plates</td>
 <td>Becton Dickinson</td>
 <td>353046</td>
 <td></td>
 </tr>
 <tr>
 <td>1.8 ml CryoTube vials</td>
 <td>Nalgene Nunc</td>
 <td>377267</td>
 <td></td>
 </tr>
 <tr>
 <td>Controlled Rate Freezer</td>
 <td>Thermo</td>
 <td>CryoMed</td>
 <td></td>
 </tr>
 </tbody>
</table>

preparation of tumor antigen-loaded mature dendritic cells for immunotherapy

Klinik çalışmalarda antijen yüklü DC aşıları güvenli ve tümörler 1 için umut verici tedavi olduğunu kurduk da, klinik etkinliği kurulacak kalır. Bir yöntem olup, İyi Üretim Prosesi (GMP) kurallarına uygun olarak hazırlanan, aşağıda açıklanan klinik çalışmalar 2 DC&#39;lerin sayıda üretmek için en yaygın ex vivo olarak hazırlama yönteminin bir iyileştirmedir. Bizim yöntem sentetik TLR 3 agonisti DC uyarmak için Polyinosinic-polisitidilik Asit-poli-L-lisin Karboksimetilselüloz (Poli-ICLC) kullanır. Daha önceki çalışmada, CD83 ve CD86, interlökin-12 indüksiyonu (IL-12), tümör nekroz faktörü (TNF), interferon gama-kaynaklı bir regülasyonu ile değerlendirildi gibi poli-ICLC insan DC&#39;ler için en güçlü tek olgunlaşmasını teşvik olduğunu tespit Protein 10 (IP-10), interleukmin 1 (IL-1) ve tip I interferonlar (IFN), ve en az interlökin 10 (IL-10) üretimi. </p&gt; DC lökaferezis ile elde edilen dondurulmuş periferik kan mononükleer hücreleri (PBMC) ayrılıyor. PBMC, Ficoll gradyan santrifüj ile izole edildi ve alikotlar halinde dondurulur. 1. günde, PBMC çözülmüş ve ° C doku kültürü inkübatöründe 37 de 1-2 saat inkübasyondan sonra plastik yüzeye yapışma monositler seçmek için doku kültürü şişeleri üzerine kaplanmıştır. İnkübasyondan sonra, lenfositler yıkanır ve yapışık monositler interlökin-4 varlığında (IL-4) ve olgunlaşmamış DC&#39;lerin ayırt etmek için, granülosit-makrofaj koloni uyarıcı faktör (GM-CSF), 5 gün boyunca kültürlenmiştir. 6. günde, olgunlaşmamış DC&#39;lerin aşı kalitesi için bir kontrol olarak hizmet eder ve aşı 3 immünojenisitesini artırmak olabilir anahtar deliği limpet hemosiyanin (KLH) proteini ile pals edilmiştir. DC&#39;ler olgunlaşması için uyarılmış peptit antijen ile yüklenir ve bir gece inkübe edilir. 7. günde, hücreler yıkandı ve ihtiva eden 1 ml&#39;lik kesirler dondurulur4 - kontrollü bir sınıf dondurucu kullanarak 20 x 10 6 hücre. DH partiler için çok sürümü test yapılır ve hasta enjekte edilmeden önce minimum teknik özellikleri karşılamalıdır.

10 arası - Başlangıç ​​PBMC&#39;ler% 20, kültür periyodunun sonunda DC&#39;ler farklılaşırlar. Olgun DC CD11c olan +, CD14-, CD83 +, CD40 + ve CCR7 + (Şekil 1). Bunlar MHC sınıf I ve sınıf II molekülleri ile birlikte uyaran moleküller CD80 ve CD86 arasında yüksek düzeyde ifade eder. Diğer TLR agonistleri 14 ile karşılaştırıldığında, poli-ICLC da PDL-1 daha düşük düzeyde arttırmaktadır. Ayrıca, bu Poli-IC-olgunlaşmış DC&#39;ler salgılar büyük IL-12 ...

Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Tümör immünoterapide kullanım için otolog dendritik hücreler, çok sayıda (DC&#39;ler) oluşturmak için en yaygın olarak kullanılan yöntem tarif edilmektedir. Gibi bir yöntem monositlerden DC&#39;ler ayırt etmek için, IL-4 ve GM-CSF kullanır. Bu hastaya geri enjekte edilmeden önce olgunlaşmamış DC&#39;lerin olgun ve daha sonra antijen ile yüklenir için uyarılır.

İmmünoterapi için Tümör Antijen-yüklü Olgun dendritik hücreler hazırlanması

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

preparation-tumor-antigen-loaded-mature-dendritic-cells-for

Research

JoVE Journal

Immunology and Infection

18.1K Görüntüleme.  NYU Langone Medical Center. Tümör immünoterapide kullanım için otolog dendritik hücreler, çok sayıda (DC'ler) oluşturmak için en yaygın olarak kullanılan yöntem tarif edilmektedir. Gibi bir yöntem monositlerden DC'ler ayırt etmek için, IL-4 ve GM-CSF kullanır. Bu hastaya geri enjekte edilmeden önce olgunlaşmamış DC'lerin olgun ve daha sonra antijen ile yüklenir için uyarılır.

Video: Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Murin Model B hücreleri CD40 aktivasyonu

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

İmmünoloji ve Enfeksiyon

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Hücre olgunlaşması olmadan Dendritik Hücreler Verimli Transfeksiyon için optimize edilmiş Protokolü

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Fare Akciğer Dendritik Hücrelerinin İzolasyonu

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

İnsan Thymus ile myeloid dendritik hücreleri ve epitel hücreleri İzolasyonu

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Bakım Glioblastoma Standardı Kapsamında Adoptif Tedavisi için CAR T hücrelerinin üretilmesi

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Metabolik fenotipleri farklı olarak sahip Olgunlaşmamış, Olgun ve tolerojenik Dendritik Hücrelerinin Üretimi

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

ile Enfeksiyon sırasında boşaltılması Lenf düğümleri içine Fare Cilt Dendritik Hücre Göç Eğitim Bir KAKE tabanlı Testi<em&gt; Mycobacterium bovis</em&gt; Basil Calmette-Guerin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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

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

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

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

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Geliştirme ve doğrulama vericiyi bir tek molekül dijital enzim bağlı Immunosorbent Assay insan Interferon-α için dizi

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Fizyoloji Immunogenic dendritik hücreler oluşturma için yeni protokol

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...