Proje NIH NIAID Sözleşme No HHSN2662000500021C tarafından desteklenmiştir. Ming Chen Biz onun teknik destek için teşekkür ederiz.

1. Programı Amaxa 96 servis Nucleofector <ol><li> Yeni bir parametre dosyasını açın. </li><li> 96 plaka şeması üzerine imleci sürükleyerek standart transfeksiyon için kullanıyor olacak kuyuların sayısını seçin. Havuza her bir deney numune için en az 3 kuyu kullanın. </li><li> Giriş program kodu: part1 seçin &#39;FF&#39; ve part2 seçin &#39;168 &#39;aşağı çekme menüler </li><li> Çözüm kutusunu seçin &#39;Monosit, insan&#39; </li><li> Kontrol Seçeneği altında &#39;Standart&#39; seçeneğini seçin. </li><li> Uygula tıklayın. </li><li> Transfeksiyon kontrolü için gereklidir ve daha sonra Kontrol Seçenek &#39;No Program Kontrol seçin ve Uygula tıklayın şemada daha kuyuları seçin. </li><li> Plaka diyagram üzerinde kalan kullanılmayan kuyu seçin ve undefine üzerine tıklayın. </li></ol> 2. DC&#39;ler transfeksiyon için hazırlayın <ol><li> Tüm çalışmalar, mümkünse bir hücre kültürü kaputu steril koşullar altında yapılmalıdır. Ekleyerek nucleofection çözüm hazırlayın 96-450 2025 oranında İnsan Monosit 96-iyi Nucleofector Çözüm için ek. Mix ve oda sıcaklığına kadar ısınmasını bekleyin. Ortalama nucleofection çözüm 20μl gerek. % 10 aşırı hata pipetleme için izin olun. </li><li> 1 ve 2 satır ilki takmadan doğru yönde yani nucleocuvette plakasına ihtiyaç nucleocuvette modülleri sayısı yerleştirin. </li><li> Deney, 10 dakika süreyle 400g santrifüj iyi ve pelet ortalama 500.000 hücreleri hesaplamak için gerekli DC&#39;ler sayısını belirler. Süpernatantı dikkatlice çıkartın. </li><li> Hafifçe birkaç kez aşağı yukarı pipetleme ve nucleofection çözüm hücrelerin tekrar </li><li> Yeniden süspanse hücreleri belirli bir tedavi örneğin GLO ve Rig-I etiketli eppendorfs içine doğru miktarda bölün. </li><li> Pipetleme 500.000 hücreleri ve karışım başına 0.25μg siRNA ekleyin. No-transfeksiyon kontrol örneği olmayan hedef siRNA kullanın. </li><li> Deneme düzeni göre nucleocuvette modüller halinde yukarıdaki karışımları Pipet 20μl, sıvı sağlayan kuyunun dibine teslim edilir. </li><li> Kapaklı nucleocuvette plaka Kapak ve hava kabarcıklarının çıkarılması yardımcı olmak için birkaç kez sert bir yüzeye plaka dokunun. </li></ol> 3. Transfect DC&#39;ler <ol><li> Plaka Nucleofector 96-iyi bir servis tepsisine yerleştirin Yükle tıklatın ve sonra başlayabilirsiniz. </li><li> Lap top ekranda transfeksiyon sürecinin ilerlemesini takip, kırmızı zemin üzerine siyah bir çubuk başarısız olduğu anlamına gelir, oysa yeşil bir arka plan üzerinde siyah haç, çok iyi başarılı bir transfeksiyon anlamına gelir. </li><li> Transfeksiyon sürecinin tamamlanmasından sonra, plaka kaldırmak ve her biri de çok kanallı pipet kullanarak DC büyüme orta 80μl ekleyin. </li><li> 37 ° 10 dakika plaka ° C&#39;de ve% 5 CO 2. </li><li> Doğru yönünü koruyarak, nucleocuvettes 100μl hacmi, önceden ısıtılmış DC büyüme orta 100μl içeren matris tüpler içine aktarın. </li><li> Transfeksiyon meydana gelmedi bu tüpleri çıkarın ve atın. </li><li> 37 ° ° C&#39;de ve% 5 CO 2, 24 saat veya diğer istenilen zaman aralığı için . </li></ol> 4. NDV ile hücreleri enfekte <ol><li> Eppendorfs her deneysel örnek inkübatör hücre kültürü kaput ve havuz tüpleri matris tüpleri çıkarın </li><li> Pelet hücreler yavaşça 5 dakika için bir masa üstü santrifüj iplik ve süpernatant kaldırmak. </li><li> 1 bir İçişleri Bakanlığı NDV içeren serum büyüme ortamda hücrelerin yeniden süspanse edin ve 37 inkübe ° C ve gevşek steril bir moda kapalı eppendorfs% 5 CO 2, 45 dk. </li><li> 8-10 saat DC büyüme orta 900μl ve yeniden inkübe ekleyin </li></ol> 5. Hasat hücreleri <ol><li> Iplik ve bir masa üstü santrifüj eppendorfs süpernatant kaldırmak Pelet hücreleri. </li><li> RNA ve protein izolasyonu protokole göre Hasat hücreleri. </li></ol> 6. Temsilcisi sonuçları: Bizim optimize protokolünü kullanarak DC&#39;ler transfekte Rigi-hedef interferon yanıtı yol uyarmak için NDV (RIG-I tarafından tespit Paramyxovirus) ile hücreleri enfekte sonra 24 saat siRNA ve. QRT-PCR analizi biz gen transkripsiyon seviyesinde% 75 oranında yıkmak gösterdi. Biz de IFN sinyalizasyon kaskad RIG-I downstream efektör IFNβ ifade benzer bir azalma gözlemledik. Ayrıca, MXA IFNβ mansap yanıt gen, (Şekil 1A) minimum iken enfekte olmayan, kontrol transfekte hücrelerinde IFNβ ifade algılanamaz olduğunu gözlemledik. Ile DC&#39;ler ikinci bir transfeksiyon Rigi hedef siRNA Western blot analizi için yeterli hücre kullanılarak yapıldı. NeredeRT-PCR sonuçları ile benzer bulunmuştur (yıkmak RIG-I ve IFNβ ifade sırasıyla% 62 ve% 66) daha önce gördüm (Şekil 1B), Western Blot bu gen ekspresyonu ortaya konmuştur I-Rig probed (Şekil 1C) tamamen engelledi. <img src="/files/ftp_upload/2766/2766fig1.jpg" alt="Şekil 1A"/> Şekil 1. (A) MoDCs ya siRNA hedef ile transfekte Rig-I veya QRT-PCR ile tespit Rig-I, IFNβ ve MXA ifade nonspesifik GLO siRNA ve etkisi. (-) Transfeksiyon protokolü monosit özel bir tampon ve nucleoporation programı FF168 (Lonza Walkersville A.Ş.) 24 saat süreyle takiben kuluçka transfekte hücreler ya NDV (+) veya sol bulaşmamış ile enfekte edildi. 10 saat süreyle bir başka inkübasyondan sonra, hücreler toplandı ve RNA ekstrakte Transkript seviyeleri iki çoğaltmak deneylerin sonuçlarını temsil edildi. Bowles ve ark 16 alınan (B) Şekil 1A için kullanılan farklı bir buffy coat elde MoDCs tekrar ile transfekte ya Rig-I-siRNA veya nonspesifik GLO siRNA Yukarıdaki gibi aynı transfeksiyon protokolü kullanılarak hedef. Untransfected MoDCs kullanan bir ek kontrol deneyi içine dahil oldu. 24 saat inkübasyondan sonra tüm hücreleri NDV ile enfekte ve RNA ve protein izolasyonu için hasat edilmeden önce bir 10 saat daha inkübe edildi. Rig-I IFNβ QRT-PCR ile belirlenen MXA transkript seviyeleri iki çoğaltmak deneylerin sonuçları temsil eder. Bowles ve ark 16 Taken (C) Şekil 1B de yukarıda açıklanan hücrelerden lizatları Western blot ile analiz edildi. Untransfected hücreleri lizatları Lanes 1 ve 2, 3 ve 4 şeritli GLO siRNA transfekte hücreler lizatları şeritleri 5 ve 6, Rig-I-hedef siRNA transfekte hücreler lizatları. Örnekler RIG-I probed ve aynı zamanda GAPDH (bir yük kontrolü gibi) ve iki nüsha halinde çalışır. Bowles ve ark 16 alınmıştır.

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Verimli naif birincil dendritik hücrelerin transfeksiyon, yüksek verimlilik analizi ve, doğuştan gelen adaptif bağışıklık geçiş aracılık bu anahtar hücre, hücresel inflamatuar yollarının tersine mühendislik için önemlidir. Ancak, çoğu araştırmacı bu hücrelerin standart transfeksiyon teknikleri kullanarak hem verimli ve transfeksiyon prosedürü inducing hücre olgunlaşması olmadan transfect zor olduğunu bulabilirsiniz. Bu sınırlamalar, eş zamanlı, bağımsız bir 96-iyi ticari nükleer transfeksiyon sistemi (Lonza) kullanarak yüksek verim protokol optimizasyonu ile aşılabilir olmadığını araştırdık. Floresan aktif hücre sıralama (FACS) kullanarak, GFP ifade transfeksiyon verimliliği ve bir hücrenin olgunlaşma okuma olarak CD86 ve MHCII immünoreaktivite bir marker olarak plazmid ölçülür. Sonuçta&gt;% 50 transfeksiyon ve olgunlaşma belirteçlerinin artış olmaması gösteren koşullar belirlenmiş tamponlar ve elektroporasyon programlarının değerlendirilmesi deneylerin bir dizi. Nucleofection süreci daha DC&#39;ler mümkün aktivasyonu araştırmak için mRNA düzeyinde IFNβ ekspresyon düzeyleri ve onun downstream tepki gen MXA analiz MXA ifade IFNβ üretim zarif hassas ve çok düşük seviyelerde tespit etmek için bir biyoassay olarak kullanılabilir cytokine18. Mah-PCR analizi sayesinde saptanabilir IFNβ ifade gördüm ama MXA böylece yukarıda bazal IFNβ indüksiyon gösteren bazı upregülasyonu gördün. Ancak enfekte olmayan, kontrol transfekte hücrelerinde MXA bu minimal upregülasyonu IFNβ sinyal kaskad (Şekil 1A) virüs aracılı stimülasyon kaynaklanan kıyasla yok denecek kadar azdı ve başarıyla aktive olmadan DC&#39;ler transfect nucleoporation protokolünü kullanmak doğruladı herhangi bir anlamlı düzeyde. Bizim optimize protokol programı Western Blot analizi ile teyit edilmiştir. SiRNA hedef Rig-I saptanabilir Rig-I protein (Şekil 1C) tam bir kaybına neden oldu. DC&#39;ler Transfeksiyon Biz diğer genlerin başarılı pertürbasyon, siRNA miktarı ve transfeksiyon zaman uzunluğu optimize edilmesi olabilir dikkat etmelisiniz. Bildiğimiz kadarıyla, bu çalışmanın, böylece bu alanda araştırma için zor bir engel çözme ilk kez primer insan DC&#39;ler yüksek verimlilik kaybı fonksiyon testleri tasarım sağlar. Bu DC sinyal çalışma için yeni fırsatlar sunmak ve DC-tabanlı Tedaviler geliştirme ilerlemesine katkıda bulunabilir.

<table border="1"><tbody><tr><th> Ekipman / reaktif </th><th> Şirket </th><th> Katalog # </th><th> Yorumlar </th></tr><tr><td> Amaxa Nucleofector 96-iyi Servisi </td><td> Lonza </td><td> 108S0109 </td><td> Seri numarası </td></tr><tr><td> Amaxa İnsan Monosit 96-Nucleofector Kiti </td><td> Lonza </td><td> VHPA-2007 </td><td> İnsan monosit 96 Nucleofector Çözüm, 96-iyi Supplement ve Nucleocuvettes ve plakalar içerir </td></tr><tr><td> Rig-I siRNA </td><td> Dharmacon </td><td> L-012.511-00 </td><td></td></tr><tr><td> GLO siRNA </td><td> Dharmacon </td><td> D-001600-01-20 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875 </td><td> Ile desteklenmiş% 10 FCS, 2 mM L-glutamin, 100 U / ml penisilin ve DC besi yapmak için 100 mg / ml streptomisin </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td><td></td></tr><tr><td> L-glutamin </td><td> Invitrogen </td><td> 25030081 </td><td></td></tr><tr><td> Penisilin / streptomisin </td><td> Invitrogen </td><td> 15070063 </td><td></td></tr><tr><td> Fetal buzağı serumu </td><td> HyClone </td><td> 3070,03 </td><td></td></tr><tr><td> Dendritik Hücreler </td><td> New York Kan merkezi </td><td></td><td> DC&#39;ler standart bir prosedür kullanılarak buffy kat arınmış. </td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

Dendritik hücreler (DC), bağışıklık sisteminin başlatılması ve enfeksiyon 1 yanıt kritik bir rol oynayan nöbetçi olarak kabul edilebilir . Naif DC&#39;ler tarafından patojenik antijen tespiti, patojen ilişkili moleküler desenleri (PAMPS) olarak adlandırılan özel korunmuş yapıları tanıyamaz örüntü tanıma reseptörleri (PRRs) geçer. DC&#39;ler tarafından PAMPs Algılama olgun DC&#39;lere aktivasyon ve dönüşüm sonuçlanabilecek bir hücre içi sinyal basamaklarını tetikler. Bu süreç genellikle diğer proinflamatuar sitokinlerin birlikte, tip 1 interferon üretimi ile karakterize, olgun T hücreleri ile etkileşim adaptif immün yanıt 2 başlatır drenaj lenf düğümleri, DC MHCII ve CD86 ve göç gibi hücre yüzey belirteçlerinin upregülasyonunun 3. Bu nedenle, DC&#39;ler doğal ve adaptif bağışıklık sistemi bağlantısı. Çeşitli patojenler DC tepkisi altında yatan moleküler ağlar incelemek için yetenek bu sinyal yollarının düzenlenmesi ve kaynaklı genlerin daha iyi anlaşılması için çok önemli. Bu aynı zamanda bulaşıcı hastalıklar ve tümörlere karşı DC tabanlı aşılar gelişimi kolaylaştırmak için yardımcı olacaktır. Ancak, bu araştırma çizgi ciddi zorluk birincil DC&#39;ler 4 transfecting engellemiştir. Virus lentiviral sistemi olarak iletimi yöntemleri, tipik olarak kullanılır, ancak bu karmaşıklık ve biyo-tehlikeli bir risk (ilgili maliyetler) 5,6,7,8 gibi birçok sınırlamalar taşır. Ayrıca, viral gen ürünleri teslim DC&#39;ler 9,10,11,12 transduced immünojenite artar. Elektroporasyon karışık sonuçlar 13,14,15 ile kullanılan, ancak biz, yüksek verimli bir transfeksiyon protokol kullanım raporu ve kesin kendi programını göstermek için ilk olmuştur. Bu yazıda, sınırlı hücre toksisitesi ve DC olgunlaşma (16) yokluğu, insan birincil DC&#39;ler yüksek verim transfeksiyon için optimize edilmiş bir ticari protokol özetleyebiliriz . Transfeksiyon verimlilik (plazmid GFP) ve hücre canlılığı fazla sırasıyla% 50 ve% 70 idi. QRT-PCR IFNβ hiçbir upregülasyonu gösterdi FACS analizi, transfekte hücrelerde olgunlaşma belirteçlerinin CD86 ve MHCII ifade artış olmaması kurdu. Bu elektroporasyonu protokolü kullanarak, başarılı transfeksiyon DC&#39;ler siRNA ve etkili hedeflenen gen yıkmak için kanıt sağlamak Rig-I mRNA ve protein seviyeleri hem de kilit bir virüs tanıma reseptör 16,17,.

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Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Biz plazmid DNA hücresinin olgunlaşması neden olmaksızın veya siRNA birincil insan monosit türevli dendritik hücreler transfecting etkin bir yolu olarak optimize edilmiş yüksek verimli nucleofection protokol sunuyoruz. Biz daha başarılı bir şekilde hedef gen, mRNA ve protein seviyeleri hem de Rig-I siRNA susturulması için kanıt sağlar.

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 ...

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JoVE Journal

Immunology and Infection

20.3K Görüntüleme.  Mount Sinai School of Medicine. Biz plazmid DNA hücresinin olgunlaşması neden olmaksızın veya siRNA birincil insan monosit türevli dendritik hücreler transfecting etkin bir yolu olarak optimize edilmiş yüksek verimli nucleofection protokol sunuyoruz. Biz daha başarılı bir şekilde hedef gen, mRNA ve protein seviyeleri hem de Rig-I siRNA susturulması için kanıt sağlar.

Video: Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

İzleme Araştırmaları Qdot nanokristaller murin Kemik İliği-türevli dendritik hücrelerin üretilmesi ve Etiketleme

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

İmmünoloji ve Enfeksiyon

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

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

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

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

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 ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Alerjik Havayolu Enflamasyon sırasındaki solunum Dendritik Hücre olgunlaşması ve Göç Analizi

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

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 ...

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 ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Nucleofection İnsan THP-1 Makrofagların Yüksek Verimli Transfeksiyonu

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Fare Dendritik Hücre Alt Grupları İzolasyonunda bir Verimli ve Yüksek Verim Yöntem

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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. ...

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.

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 ...