Bianca MOTHE Carla Oseroff ve Marie-France DelGuercio immünolojik testleri ve fare kullanımı için beni tanıştırdığı için teşekkür etmek istiyorum. GA Splitter laboratuarda çalışma NIH hibe 1-RO1-AI-073.558, GLRCE hibe 1-U54-AI-057.153, BARD ABD-3829-06 tarafından finanse edilmektedir.

1. Hedef Özel Efektör CTL&#39;lerin Bağışıklama ortaya çıkartılması (Gün 0) <ol><li> Hedef sistemi: başlamadan önce, belirli bir efektör üzerine karar verebilir. Klasik bir in vivo CTL tahlil Bu örnekte, spesifik CD8 + T hücreleri ortaya çıkarmak için saflaştırılmış bir peptid bağışıklama kullanabilirsiniz. Hedef sistemi: Aynı peptid singeneik hedef hücreleri belirli bir efektör yaratarak darbe olacaktır. Alternatif olarak, özel etkileyiciler ve hedefleri elde etmek için tüm patojen veya protein kullanmayı tercih edebilir. </li><li> Enjeksiyonları kısırlık sağlamak için çalışma yüzeyinin bir biyogüvenlik kaput içinde dekontamine. İlk 50 mg, 2 mL tüp içinde yer alan PBS / fare 100 mcL% 10 DMSO peptit saflaştırılmış koyarak peptid bağışıklama hazırlayın. Daha sonra, karıştırma için 1 mm boncuklar ile 0,2 mL işareti ekleyin ve çok soğuk Eksik Freund adjuvan bir eşit miktarda. Bu açıdan bakıldığında, enjeksiyon kadar buz üzerinde immünojen tutmak. Parafilm ile güvenli kapak. </li><li> Derhal karıştırıldıktan sonra buz üzerinde emülsiyon yerleştirerek, 3 dakika boyunca emülsiyon karıştırmak için maksimum RPM&#39;ler ayarlanmış bir boncuk çırpıcı kullanın. Emülsiyon kıvamda mayonez benzer olmalıdır. Emülsiyon 4 oturup ° C gecede herhangi bir ayrılık olduğunu kaydetti zaman izin verir (bir hafta), daha fazla karıştırma. Sağlayın </li><li> Herhangi bir boncuk yukarı çekmeyin dikkatli olmak emülsiyon çekmek için 1 ml şırınga ve 20 veya 20.5 iğne kullanın (boncuklar, iğne, ölçü daha büyük olabilir, ama yine de farkında olmak gerekir). </li><li> Subkutan enjeksiyon için kuyruk dibinde Güvenli fare. Biz, güvenli bir şekilde modifiye edilmiş bir plastik 50 ml konik tüp kullanmak ve yavaşça enjeksiyon için fare hareketsiz. 0,2 ml emülsiyon fare ile enjekte edilir. </li></ol> 2. Hasat Hedef Hücreler (Gün 8) <ol><li> Hedef hücreleri için saf singeneik fareler gerekecektir. AAALAC standartlara göre saf fareler Euthanize. </li><li> Fare, fare saç veya diğer harici kirleticiler kontaminasyon riskini en aza indirmek için% 70 Etanol püskürtülerek dezenfekte edin. </li><li> Diseksiyon makas kullanarak farenin sol tarafında deri yoluyla küçük snip olun. Periton kesesi ve iç organlarda ortaya çıkarmak için flep üzerinden çekin. </li><li> Periton yoluyla Snip, dikkatli olmanın temel organlarının herhangi bir taviz vermemek. Dalak, yüzeye yakın ve renkli karaciğer (koyu kırmızı) &#39;e benzer ama daha küçük ve şekil olarak daha barbunya. Enfekte fareler genellikle enfekte olmamış kontrollere göre özellikle büyük dalak göstermektedir. </li><li> Şekilde maksimum hücre sayıları kurtarmak için çoklu parçalar halinde dalak kırmak için nazik olmaya özen, dalak dikkatlice dışarı çekin. Hemen buz üzerinde hazırlanmış medya 10 mL içeren 15 ml konik dalak yerleştirin. Kalan fare ile devam edin. </li><li> Cam bir doku değirmeni içine 15 ml konik tüp (medya ve dalak) içeriğini dökün. Üst kol gücü bol kullanarak dalak Grind. Tüm doku rengini kaybetti bağ dokusu hücreleri kurtarıldı olduğunu varsayabiliriz. </li><li> 70 mikron hücre filtresi aracılığıyla değirmeni içeriği 50 ml konik bir tüp içine dökün. Değirmeni taze medya ile durulayın ve bir hücre süzgeç vasıtasıyla dökün. </li><li> Santrifüj 4 ° C, 7 dakika için 1300 RPM. Süpernatant kapalı dökün. </li><li> 10 mL reaktif ve 7 dakika 37 ° hazırlanan parçalama inkübe süspanse edin. </li><li> 50 mL kadar taze medya ve santrifüj, yukarıdaki gibi aynı ekleyin. </li><li> 50 mL taze medya ve santrifüj, yukarıdaki gibi aynı (yıkama adım) süspanse edin. * Not: Şu anda saf bir hedef hücre popülasyonunun transferi için alıcı farelere isteniyorsa, belirli bir hücre tipi için bir manyetik hücre ayırma (yani makrofajlar) gerçekleştirebilirsiniz. Ayrıca, şu anda hücre öldürme tedavisinin etkilerini araştırmak için deneysel bir tedavi kültürü haline getirebilir. </li><li> 96 iyi yuvarlak alt doku kültürü tedavi plaka 100μL x 10 7 de ortalama hücreleri ve plaka 1 güvenin. * Bu hücre / iyi bir yüksek konsantrasyon, ancak hızlı bir tahlil, çünkü biz bu konsantrasyonda hücre canlılığı kaybı gördük. </li></ol> 3. Hedef Hücre Tedavi ve Etiketleme <ol><li> Darbe uygun hücreleri ve patojen spesifik peptid veya alakasız peptid kontrolü ile çoğaltır: 1 mcg / ml peptid ekleyin. 1.5 saat 4 ° C&#39;de inkübe edin. Son 30 dakika 37 ° C inkübatör taşıyın. Bu süre zarfında KAKE reaktif hazırlamak. </li><li> KAKE üretici özelliklerine göre hazırlayın ve 0.5 ile 25 mcM arasında bir çalışma konsantrasyonu sulandırmak. &quot;Düşük&quot; konsantrasyon &quot;yüksek&quot; konsantrasyon KAKE daha düşük 10x olun. Örneğin, [az] 10 mL PBS 0.5 mcL KAKE ekleyin ve [yüksek], 10 mL PBS 5 mcL KAKE eklemek. Sıcak hazırlanan KAKE 37 ° C etiketleme önce. </li><li> Kuvöz ve p hücreleri plaka çıkarınellet, oda sıcaklığı, 7 dakika için 1300 RPM yüksek hızda santrifüj. Süpernatant kaldırmak Flick. </li><li> [Düşük] veya ilgili kuyulara uygun reaktif 200 mcL ekleme ve 37 az 15 dakika boyunca inkübe ° [yüksek] KAKE C ya da yeniden süspanse hücreleri </li><li> Oda sıcaklığında, 7 dakika için 1300 RPM, kuvöz ve tekrar pelet hücreleri plaka çıkarın. Süpernatant kaldırmak Flick. </li><li> 200 mcL / iyi ısıtılmış taze medya ve 30 dakika boyunca inkübe yeniden süspanse hücreleri 37 ° C </li><li> Oda sıcaklığında, 7 dakika için 1300 RPM, kuvöz ve pelet hücreleri plaka çıkarın. Süpernatant kaldırmak Flick. </li><li> 100 mcL / iyi PBS içinde süspanse edin hücreleri. </li><li> Evlatlık transferi için, bir de [az] biri etiketli hücreleri [yüksek] fare başına düşen hücreler etiketli. Enjeksiyon sonuçlanan 0,2 mL% 50 belirli hedefleri ve% 50 ilgisiz hücreleri olmalıdır. </li><li> (Bakınız Şekil 1), veri analizi sırasında ve akış sitometresinde çalışan olmayan transfer kontrolleri olarak hareket etmek bazı etiketli hücreler bir kenara bırakarak unutmayın. </li></ol> 4. Evlatlık transferi <ol><li> AAALAC standartlarına göre önceden peptid aşı fareler anestezi (part # 1 Bu protokolün). </li><li> 0,2 mL alıcı farelere retroorbital yolla etiketli hedef hücreleri enjekte edilir. </li><li> Alıcı fareler euthanizing 6 saat önce ya da diğer önceden belirlenmiş en uygun zamanı bekleyin. </li></ol> 5. Hasat Etiketli Hücreler * Not: 5.1 5.11 üzerinden 2.11 üzerinden 2.1 adımları aynı Adımlar <ol start="12"><li> 96 yuvarlak alt doku kültürü tedavi plaka 100 mcL x 10 6 sıra ortalama 1 hücreleri ve plaka dışarı güvenin. </li><li> Tüm kuyuların% 1 paraformaldehid nihai konsantrasyonu 100 mcL% 2 paraformaldehid ekleyerek kurtarıldı ve non-transfer hücreler sabitleyin. </li></ol> 6. Akım Sitometri ve Veri Analizi <ol><li> Bir akış sitometresinde örnekleri çalıştırın ve verileri analiz. </li></ol> 7. Temsilcisi Sonuçlar Ortaya çıkan histogram azalan epitop özel zirve hücre belirli bir öldürme (Şekil 1) bir göstergesi. Hücre özel lizis gerçek değerini belirlemek için aşağıdaki denklemler (Tablo 1 uygulanan) kullanın: Oranı = Alakasız yüzdesi: epitopu Özel Yüzde ([düşük] zirve: [yüksek] pik), Yüzde Özel Lizis = [1 - (Sigara transfer kontrolü oranı / Deneysel oranı)] x 100 <img src="/files/ftp_upload/2250/2250fig1.jpg" alt="Şekil 1" /> Şekil 1. BALB / c fare dalak kurtarıldı. Numaraları KAKE profili yüksek ya da düşük KAKE fenotipi hücrelerinin yüzdesini göstermektedir. A) kontrol Sigara transfer. [Düşük] KAKE doruklarına [yüksek] KAKE doruklarına ilgisiz peptit ile darbeli hedefleri temsil ederken, epitop belirli hedef hücreleri temsil eder. B) KAKE etiketli hücreler PBS aşı alıcı fare (Kontrol) kurtarıldı. C) iki farklı peptid özel alıcı fareler kurtarıldı KAKE etiketli hücreleri, azalan epitop özel doruklarına unutmayın. <img src="/files/ftp_upload/2250/2250fig2.jpg" alt="Şekil 2" /> Şekil 2. Farelerin her grup, PBS, Peptid A ve Peptid B. gösterilen analiz verileri grafiksel gösterimi üç veri noktaları <table border="1"><tbody><tr><td> Örnek </td><td> KAKE YÜKSEK% epitop Pulsed </td><td> Alakasız KAKE DÜŞÜK% </td><td> Oranı Düşük Yüksek </td><td> Yüzde epitop Özel Killing </td></tr><tr><td> PBS </td><td> 42,8 </td><td> 57,2 </td><td> 1,34 </td><td> 16,97 </td></tr><tr><td> PBS </td><td> 43,1 </td><td> 56,9 </td><td> 1,32 </td><td> 15,94 </td></tr><tr><td> PBS </td><td> 44,3 </td><td> 55,7 </td><td> 1,26 </td><td> 11,74 </td></tr><tr><td> Peptid A </td><td> 39,7 </td><td> 60,3 </td><td> 1,52 </td><td> 32,56 </td></tr><tr><td> Peptid A </td><td> 38,6 </td><td> 61,4 </td><td> 1,59 </td><td> 35,61 </td></tr><tr><td> Peptid A </td><td> 40,6 </td><td> 59,4 </td><td> 1,46 </td><td> 29,99 </td></tr><tr><td> Peptid B </td><td> 38,7 </td><td> 61,3 </td><td> 1,58 </td><td> 24,68 </td></tr><tr><td> Peptid B </td><td> 38,5 </td><td> 61,5 </td><td> 1,60 </td><td> 25,32 </td></tr><tr><td> Peptid B </td><td> 39,3 </td><td> 60,7 </td><td> 1,54 </td><td> 22,76 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> PBS NT Control </td><td> 47,4 </td><td> 52,6 </td&gt; <td> 1,11 </td><td></td></tr><tr><td> Pep bir NT Kontrol </td><td> 49,4 </td><td> 50,6 </td><td> 1,02 </td><td></td></tr><tr><td> Pep B NT Kontrol </td><td> 45,6 </td><td> 54,4 </td><td> 1,19 </td><td></td></tr></tbody></table> Tablo 1. Temsilcisi veri analizi. &quot;NT&quot; olmayan aktarılır.

<ol>
	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

Çıkar çatışması ilan etti. Wisconsin-Madison Üniversitesi Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

KAKE döl 10,11 arasında nispeten eşit böler, çünkü bu testte, klonal genişleme de dahil olmak üzere hücrelerin proliferatif kapasitesi araştırmak için değiştirilebilir. Örneğin tetramers ve biyoparlaklık 13, KAKE etiketleme ile kombine edilebilir hipoteze göre daha uygun olabilir yöntemleri diğer hücre izleme, var olmasına rağmen, hücre göçü 6,12 araştırmak için KAKE etiketleme kullanmak da mümkün. PE de işe yaramayabilir ise...

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<td>1 mm glass beads</td>
<td>&nbsp;</td>
<td>Biospec Products</td>
<td>11079110</td>
<td>&nbsp;</td>
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<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
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<td>96-well plate</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>3799</td>
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<td>Adjulite Incomplete Freund's Adjuvant</td>
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<td>V12883</td>
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antigen specific in vivo killing assay using cfse labeled target cells

Carboxyfluorescein diasetat succinimidyl ester (KAKE) kolay ve hızlı bir şekilde in vivo soruşturma ilgi bir hücre popülasyonu etiket kullanılabilir. Bu etiketleme klasik çoğalması ve göç çalışması için kullanılır olmuştur. Burada sunulan yöntem, epitop KAKE etiketli hedef hücreleri 4-6 hayatta kalma ve öldürme bakmak için evlatlık transferinden sonra, zaman çizelgesi kısaltılmış var. Miktarları yanıltıcı olabilir gibi spesifik CD8 + T hücre klonu öldürme seviyesi, kalite yanıt gösterebilir. CD8 + T hücreleri, sitokin üretimi ve öldürme 7,8 düşüş zaman içinde işlevsel olarak tükenmiş olabilir Özgül. Ayrıca, belirli CD8 + T hücre klonlar TCR özgüllükleri 9 farklı yanı sıra diğerleri gibi öldürmek olmayabilir. Etkili Hücre aracılı bağışıklık (CMI), antijenler, T hücreleri yanıt yeterli sayıda sadece üreten tespit değil, aynı zamanda işlevsel olarak sağlam bir yanıt T hücreleri olmalıdır. Burada iki peptid BALB / c farelerde spesifik T hücre klonları yüzde hücre özel öldürme değerlendirir.

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Birçok enfeksiyonları güçlü bir CTL yanıtı ortaya çıkarmak, fakat bazen yanıt hücrelerinin miktarı patojenin kontrol etmek için orantılı değildir<sup&gt; 1</sup&gt;. CTL kaliteli bir önlem özellikle öldürmek için yeteneklerini<sup&gt; 2</sup&gt;. Hedef hücrelerin KAKE etiketleme bu CTL yanıt kalitesini araştırmak için kullanılabilir<em&gt; In vivo</em<sup&gt; 3,4</sup&gt;.

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.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

19.5K Görüntüleme.  University of Wisconsin-Madison. Birçok enfeksiyonları güçlü bir CTL yanıtı ortaya çıkarmak, fakat bazen yanıt hücrelerinin miktarı patojenin kontrol etmek için orantılı değildir 1. CTL kaliteli bir önlem özellikle öldürmek için yeteneklerini 2. Hedef hücrelerin KAKE etiketleme bu CTL yanıt kalitesini araştırmak için kullanılabilir In vivo

Video: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

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 ...

İmmünoloji ve Enfeksiyon

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Antijen Özgül Düzenleme için Trans-vivo Gecikmeli Tip Aşırı Duyarlılık Testi

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

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 ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Otoimmünite karşı Kök Hücre türetilmiş antijen-özgü düzenleyici T hücrelerinin gelişimini

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo Tahlil antijen spesifik T-hücre Cytolytic işlevi bir aşı modeli kullanılarak tespiti için

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Kanda dolaşan nadir antijen spesifik B hücreleri Discriminative insan monoklonal antikorların üretimi

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Bir Protein Mikroarray tahlil antijen spesifik antikor yanıt aşağıdaki Clostridium difficile enfeksiyon serolojik belirlenmesi için

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic öldürme tahlil bakteriyel patojenlere karşı immünolojik yanıt değerlendirmek için

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Antijen Özgül In Vivo Killing Testi