1. PBMC, izolasyon <ol><li> BD Vacutainer CPT Hücre Hazırlık Tüp Sodyum Heparin ile kan (7,5-8,0 ml) çizin. Tüp sodyum heparin, antikoagülan ve Ficoll Hypaque yoğunluğu sıvı, artı, iki sıvı ayıran bir polyester jel bariyer, içerir. 1800 x g&#39;de 20 dakika boyunca yatay bir rotor (dışarıya başını) oda sıcaklığında (18 ° C - 25 ° C) tüp / kan örneği santrifüjleyin . Frenler santrifüj geçin. </li><li> Santrifüj sonra, katmanlar dizisi (yukarıdan aşağıya doğru görülen) aşağıdaki gibi oluşur: a) plazma b) periferik kan mononükleer hücreleri (hücre) ve trombositler - c) yoğunluk çözüm - d) polyester jel - e) granülositler - f) kırmızı kan hücreleri (Şekil 1). </li><li> Interfaz yukarıda plazma hücre katmanı bozmadan 5 ila 10 mm bırakarak, plazma tabakasının bir kısmını toplayın. Plazma kültür (bölüm 2.5) için kullanılabilir. </li><li> Interfaz bir pipet ve 15 ml konik tüp transferi ile zenginleştirilmiş fraksiyonu (hücre) Hasat. </li><li> 10 ml PBMC yıkayın fosfat tamponlu salin (PBS), tersine tüp ile 5 kez ve daha sonra x 400 g de 5 dakika süreyle santrifüj. </li><li> Yıkama adımları iki kez tekrarlayın ve sonra 5 ml PBS içinde hücre pelletini tekrar süspansiyon haline getirin. Hücre sayısını belirler. Genellikle 6 hücreli 1,3 x10, 1 ml tam kan kurtarıldı . </li><li> Frekans ve flow sitometri (Bölüm 3) (Şekil 2) tarafından hücre γδ T hücre fenotipi belirleyin. </li></ol> 2. Γδ T hücreleri, Genişleme <ol><li> 400 x g oda sıcaklığında 5 dakika boyunca 15 ml konik tüpler hücre süspansiyonları Santrifüj ve süpernatantlar atın. </li><li> Kültür vasatı (CM), sırasıyla 1000 IU / ml ve 5 mcM son konsantrasyonlarda insan IL-2 (IL-2) ve zoledronate (Zometa) ekleyerek hazırlayın. ALyS203 (Hücre Bilim ve Teknoloji Enstitüsü) veya OpTmizer (Invitrogen) medya γδ T hücreleri (6 ve 9 referanslar daha fazla bilgi) iyi bir genişleme destek. Zometa sıvı formda (4 mg/5-ml flakon) sağlanır. 5 mcM çözelti hazırlamak için, 30 ml kültür ortamı Zometa 50 ul ekleyin. </li><li> Yeniden süspanse kültür ortamında hücre pelet ve 1x10 6 hücre / ml ayarlayabilirsiniz. </li><li> 24 plaka her kuyuya 1x10 6 hücre içeren CM pipetleyin 1 ml. Büyük ölçekli kültürleri için, hücre plaka kuyuları, tabak, ya da balonun yüzey alanlarına göre 2, 0.5 x 10 6 hücre / cm az numaralı seribaşı olabilir. </li><li> Kültür (24 plaka her bir kuyu için 100 ul) hacmi yaklaşık% 10 olduğunu otolog plazma (bölüm 1.3), toplanmış insan AB sera ya da FCS ekleyin. 37 nemlendirilmiş bir tabak koyun ° C,% 5 CO 2 kuvöz 24-48 saat. </li><li> 0.5-2 x 10 6 hücre / ml bir hücre yoğunluğu kültür koruyun. Her 2-3 günde bir insan IL-2 (1000 IU / ml) (Zometa olmadan) içeren taze orta ekleyin ve hücre proliferasyonu derecesi (Şekil 3) göre, kültür hücreleri gerektiğinde yeni kuyu veya şişeler içine aktarmak . Kaynağı plazma veya serum konsantrasyonu en az% 1 korunabilir böylece orta serum. </li><li> 12-14 gün ve sıklığı, fenotip ve flow sitometri γδ T hücreleri (aşağıya bakın) fonksiyonları belirlemek Hasat hücreleri. </li></ol> 3. Flow sitometri ile fenotipik analizi <ol><li> Floresan ile aktive olan hücre sıralama (FACS) boruları 2 x 10 5 hücre içeren 200 ul örnekleri aktarın. </li><li> 2 ml soğuk PBS ekleyin ve 5 dakika boyunca 400 x g santrifüj. Ardından, 50 ul FACS tamponu (PBS +% 1 FCS +% 0.1 sodyum azid) pelet tekrar süspansiyon haline getirin. Örnekleri (monoklonal antikorlar Tablo 1&#39;de yer alan) her antikorların 5 ul ekleyin. </li><li> 20 dakika süreyle karanlıkta buz üzerinde inkübe edin. </li><li> Her bir numune için 2 ml FACS tampon ekleyin ve sonra vorteks. 4 x 400 g ° C 5 dakika Santrifüj örnekleri Süpernatantı dikkatlice süzün. </li><li> 300 ul FACS tampon ve vorteks hücrelerin tekrar (Şekil 2) bir akış sitometresinin örnekleri analiz. </li></ol> 4. IFN-γ üretimi tahlil 10 <ol><li> RPMI 1640 orta artı Zometa (5 mcM) (bundan sonra Z DAUDI belirlenen) ile bir gecede% 10 FCS (RPMI-10) gün önce tahlil, kültür 3-5 x10 5 DAUDI hücre / ml stimülatörü hücreleri hazırlar. </li><li> 2x10 6 hücre / ml RPMI-10 Z-DAUDI ve tekrar süspansiyon toplayın. Yuvarlak bir alt 96 plaka her bir kuyu için 100, Z-DAUDI 5) (2x10 ul ekleyin . </li><li> RPMI-10 A 20 mg / ml Brefeldin içeren 2x10 6 hücre / ml γδ T hücreleri hazırlayın. Her bir kuyunun Z-DAUDI hücreleri içeren γδ T hücre süspansiyonu (2x10 5) 100 ul transferi veya kuyulardan (100 sadece RPMI-10 ul, ya da phorbol 20 ng / ml, 12-miristat 13-asetat ile RPMI- 10 kontrol[PMA] artı 2 mcg / ionomycin ml). </li><li> Birkaç kez aşağı yukarı pipetleme karıştırın. 37 ° C,% 5 CO2 inkübatör 4 saat süreyle inkübe edin. </li><li> 4 ° C&#39;de 5 dakika boyunca 400 x g plaka Santrifüj ve soğuk PBS 200 ul pelet tekrar süspansiyon haline getirin. </li><li> FACS tüpler örnekleri aktarın. 4 ml soğuk PBS ekleyin ve 4 x 400 gr az 5 dakika süreyle tüpler santrifüj ° C </li><li> FITC-konjuge anti-TCRVγ9 (5 ul) ve anti-CD3 PE/Cy5-conjugated mAb (ul 2.5) ile 50 ul FACS tampon pelet tekrar Oda sıcaklığında 15 dakika inkübe ışıktan korunmalıdır. </li><li> IntraPrep reaktif 1 100 ul ekleyin ve oda sıcaklığında 15 dakika inkübe edin. 400 x g oda sıcaklığında 5 dakika boyunca her santrifüj tüpü ve 4 ml PBS ekleyin. </li><li> Aspirasyon ile süpernatantı ve IntraPrep reaktif 2 100 ul ekleyin. Titreme olmadan oda sıcaklığında 5 dakika inkübe edin. </li><li> PE-konjuge anti-IFN-γ mAb test tüpüne 5 ul ekleyin. Oda sıcaklığında 15 dakika inkübe ışıktan korunmalıdır. </li><li> 400 x g oda sıcaklığında 5 dakika boyunca her santrifüj tüpü ve 4 ml PBS ekleyin. Aspirasyon ile süpernatantı ve FACS tampon 0.5 ml hücre pelletini tekrar süspansiyon haline getirin. </li><li> Akış sitometresinde hücreleri analiz edin. CD3 + TCRVγ9 + hücreler Kapısı ve IFN-γ (Şekil 4) ifadesidir incelemek. </li></ol> 5. Temsilcisi Sonuçlar: Hücre kültür başlangıcında γδ T hücrelerinin yüzdesini belirlemek için önemlidir. Şekil. 2 A, CD3 + TCRVγ9 yüzdesi + γδ T hücreleri, hücre içinde 0. günde% 1.6 idi . Merkezi bellek fenotipleri - baskın popülasyonları CD27 + CD45RA + naif ya da CD27 + CD45RA . Γδ T hücreleri etkin bir şekilde uyarılır, (Şekil 3 A ve B) gün 3-5 kümeler oluşturdu. Küme oluşumu gecikir, büyüme gibi diğer hücre türleri, CD4 + veya CD8 + αβ T hücreleri ve NK hücreleri γδ T hücreleri (Şekil 3 C ve D) büyüme hakim olabilir. 14 gün sonra kültür, γδ T hücrelerinin sıklığı% 93.8 başarılı γδ T hücre kültürleri (Şekil 2 E) kültür hücreleri daha fazla arttı. Kültürlü γδ T hücreleri upregüle NKG2D ve CD69 ekspresyonu (Şekil 2 G ve H). CD27 görüntülenir - CD45RA - efektör bellek fenotipi (Şekil 2 F). Sitokin üretimi ve sitotoksisiteye ilişkin γδ T hücrelerinin fonksiyonları değerlendirildi. Hücre içi IFN-γ boyama γδ T hücreleri PMA / ionomycin tedavi veya Z-DAUDI hücreleri IFN-γ üretilen olduğunu göstermiştir zoledronate tedavi sonrası biriken IPP (Şekil 4). Bu sonuçlar, bu zoledronate verimli bir şekilde uyaran ve fonksiyonel γδ T hücreleri genişletebilirsiniz göstermektedir. <table border="1"><tbody><tr><td> tüp </td><td> FITC </td><td> PE </td><td> EÇG </td><td> PE/Cy5 </td></tr><tr><td> 1 </td><td> CD3 </td><td> CD19 </td><td> CD45 </td><td> CD14 </td></tr><tr><td> 2 </td><td> CD3 </td><td> TCRαβ </td><td> CD4 </td><td> CD8 </td></tr><tr><td> 3 </td><td> CD3 </td><td></td><td></td><td> CD56 </td></tr><tr><td> 4 </td><td> TCR Vγ9 </td><td> TCRαβ </td><td> CD45 </td><td> CD3 </td></tr><tr><td> 5 </td><td> TCR Vγ9 </td><td> NKG2D </td><td></td><td></td></tr><tr><td> 6 </td><td> TCR Vγ9 </td><td> CD69 </td><td></td><td></td></tr><tr><td> 7 </td><td> TCR Vγ9 </td><td> fare IgG1 </td><td></td><td></td></tr><tr><td> 8 </td><td> TCR Vγ9 </td><td></td><td> CD45RA </td><td> CD27 </td></tr><tr><td> 9 </td><td> TCR Vγ9 </td><td></td><td> fare IgG1 </td><td> fare IgG1 </td></tr></tbody></table> Tablo 1 γδ T hücrelerinin çok renkli boyama kullanılan monoklonal antikorlar. Laboratuvarımızda yapılan γδ T hücrelerinin fenotipik analiz bir örnek Şekil. 2. <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="Şekil 1"/> Şekil 1 PBMC ayrılması. Kan (7,5-8,0 ml) BD Vacutainer CPT Hücre Hazırlık Tüp Sodyum Heparin içine çekilir ve doğrudan 1800 x g de 20 dakika süreyle santrifüj edildi. Olarak çıkan katmanları santrifüj sonra, üstten bo görüldüttom: a) Plazma - b) hücre ve trombositler - c) Yoğunluk çözüm, d) Polyester jel e) Granülositler - f) Kırmızı kan hücreleri. <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="Şekil 2"/> Şekil 2 γδ T hücreleri tipik yüzey fenotipi. PBMC 14 gün boyunca zoledronate ve IL-2 ile uyarılır. Hücreleri ile boyandı FITC etiketli anti-TCR Vγ9 ve γδ T hücreleri (A ve E) genişlemesi izlemek için anti-CD3 PE/Cy5-labeled. γδ T hücreleri TCRVγ9 kendi ifadesi ile tespit edildi ve CD27 ve CD45RA (B ve F), NKG2D (C ve G) ve CD69 (D ve H) kendi ifadesi incelenmiştir. <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="Şekil 3"/> Şekil 3 Temsilcisi γδ T hücre kültürleri. Hücre IL-2 (1000 IU / ml) ve zoledronate (5 mcM) ile uyarılır. Temsilcisi alanları (IX71 inverted mikroskop [Olympus] x 200) gösterilmiştir. Γδ T hücre kümeleri ve agrega γδ T hücreleri başarılı bir şekilde genişletilmiş 3. günde (A) ve 5. gün (B) görülebilir. Γδ T hücre büyümesi (C ve D) yeterli değildi, aksine, hiçbir kümeleri veya agrega gözlendi. <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="Şekil 4"/> Şekil 4, IFN-γ üretimi . γδ T hücreleri sadece RPMI-10 (A) ya da PMA / ionomycin (B) veya 4 saat Z-DAUDI (C) ile inkübe edildi. İlk olarak, TCRVγ9 yüzey ekspresyonu boyandı ve daha sonra IFN-γ üretimi hücre içi IFN-γ boyama ile incelendi. <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="Şekil 5"/> Şekil 5 γδ T hücre kültürü Kinetiği. (A) kültür hücrelerinin mutlak sayısı, (B) γδ T hücreleri, ve (C) belirtilen zaman noktalarında γδ T hücrelerinin mutlak sayısı yüzdesi.

<ol>
	<li>Bonneville, M., O'Brien, R. L., Born, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#947;+T+cell+effector+functions:+a+blend+of+innate+programming+and+acquired+plasticity.">&#947; T cell effector functions: a blend of innate programming and acquired plasticity.</a> Nat Rev Immunol. 10, 467-478 (2010).</li><li>Hintz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+(E)-4-hydroxy-3-methyl-but-2-enyl+pyrophosphate+as+a+major+activator+for+human+&#947;&#948;+T+cells+in+Escherichia+coli.">Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human &#947;&#948; T cells in Escherichia coli.</a> FEBS Lett. 509, 317-322 (2001).</li><li>Gober, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T+cell+receptor+&#947;&#948;+cells+recognize+endogenous+mevalonate+metabolites+in+tumor+cells.">Human T cell receptor &#947;&#948; cells recognize endogenous mevalonate metabolites in tumor cells.</a> J Exp Med. 197, 163-168 (2003).</li><li>Kabelitz, D., Wesch, D., He, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+of+gammadelta+T+cells+in+tumor+immunology.">Perspectives of gammadelta T cells in tumor immunology.</a> Cancer Res. 67, 5-8 (2007).</li><li>Roelofs, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+monocytes+are+responsible+for+gammadelta+T+cell+activation+induced+by+zoledronic+acid+through+accumulation+of+IPP/DMAPP.">Peripheral blood monocytes are responsible for gammadelta T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP.</a> Br J Haematol. 144, 245-250 (2009).</li><li>Dieli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+&#947;&#948;+T-lymphocyte+effector+functions+by+bisphosphonate+zoledronic+acid+in+cancer+patients+in+vivo.">Induction of &#947;&#948; T-lymphocyte effector functions by bisphosphonate zoledronic acid in cancer patients in vivo.</a> Blood. 102, 2310-2311 (2003).</li><li>Kondo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoledronate+facilitates+large-scale+ex+vivo+expansion+of+functional+&#947;&#948;+T+cells+from+cancer+patients+for+use+in+adoptive+immunotherapy.">Zoledronate facilitates large-scale ex vivo expansion of functional &#947;&#948; T cells from cancer patients for use in adoptive immunotherapy.</a> Cytotherapy. 10, 842-856 (2008).</li><li>Espinosa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+and+biological+activity+of+bromohydrin+pyrophosphate,+a+potent+stimulator+of+human+&#947;&#948;+T+cells.">Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human &#947;&#948; T cells.</a> J Biol Chem. 276, 18337-18344 (2001).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+profile+and+anti-tumor+effects+of+adoptive+immunotherapy+using+&#947;&#948;+T+cells+against+advanced+renal+cell+carcinoma:+a+pilot+study.">Safety profile and anti-tumor effects of adoptive immunotherapy using &#947;&#948; T cells against advanced renal cell carcinoma: a pilot study.</a> Cancer Immunol Immunother. 56, 469-476 (2007).</li><li>Murali-Krishna, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+antigen-specific+CD8+T+cells:+a+reevaluation+of+bystander+activation+during+viral+infection.">Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.</a> Immunity. 8, 177-187 (1998).</li><li>Sato, K. <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+culture+medium+on+the+expansion+of+T+cells+for+immunotherapy.">Impact of culture medium on the expansion of T cells for immunotherapy.</a> Cytotherapy. 11, 936-946 (2009).</li><li>Abe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+immunological+evaluation+of+zoledronate-activated+V&#947;9+&#947;&#948;T-cell-based+immunotherapy+for+patients+with+multiple+myeloma.">Clinical and immunological evaluation of zoledronate-activated V&#947;9 &#947;&#948;T-cell-based immunotherapy for patients with multiple myeloma.</a> Exp Hematol. 37, 956-968 (2009).</li><li>Nakajima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+study+of+adoptive+immunotherapy+for+recurrent+non-small-cell+lung+cancer+patients+with+autologous+&#947;&#948;+T+cells.">A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous &#947;&#948; T cells.</a> Eur J Cardiothorac Surg. 37, 1191-1197 (2010).</li></ol>

Çıkar çatışması ilan etti.

Burada sunulan yöntem PBMC γδ T hücrelerinin verimli genişleme sağlar. γδ T hücrelerinin etkinleştirilmesi ve geliştirilmesi zoledronate ve IL-2 sitokin üretim ve sitotoksisiteye yansıyan tam efektör fonksiyonları, geliştirmek. Bildirilmiştir sentetik phosphoantigens bromohydrin pirofosfat (BrHPP) ve 2-metil-3-butenyl-1-pirofosfat (2M3B1PP) γδ T hücreleri genişletmek, ancak ticari olarak mevcut değildir. Buna karşılık, zoledronate zaten Zometa gibi klinik uygulamalar için lisanslıdır. Bu nede...

<table border="1"><tbody><tr><th> Reaktif adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Zometa </td><td> Novartis Pharma K. K </td><td></td><td> zoledronate </td></tr><tr><td> PROLEUKIN </td><td> Novartis İlaç </td><td></td><td> rekombinan insan IL-2 </td></tr><tr><td> BD Vacutainer CPT Hücre Hazırlık Tüp Sodyum Heparin </td><td> BD </td><td> 362753 </td><td></td></tr><tr><td> RPMI1640 </td><td> Invitrogen </td><td> 21870-076 </td><td></td></tr><tr><td> ALyS203-orta </td><td> Hücre Bilim ve Teknoloji Enstitüsü </td><td> 0301-7 </td><td></td></tr><tr><td> OpTmizer </td><td> Invitrogen </td><td> 0080022SA </td><td></td></tr><tr><td> brefeldin A </td><td> Sigma </td><td> B5936-200UL </td><td></td></tr><tr><td> phorbol 12-miristat 13-asetat (PMA) </td><td> Sigma </td><td> P1585-1MG </td><td></td></tr><tr><td> ionomycin </td><td> Sigma </td><td> 13.909-1ml </td><td></td></tr><tr><td> IntraPrep </td><td> BECKMAN COULTER </td><td> A07803 </td><td></td></tr><tr><td> insan anti-CD3-FITC veya PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td> anti-insan CD4-ECD </td><td> BECKMAN COULTER </td><td> 6604727 </td><td></td></tr><tr><td> anti-insan CD8-PE/Cy5 </td><td> BECKMAN COULTER </td><td> 6607011 </td><td></td></tr><tr><td> anti-insan CD14-PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07765 </td><td></td></tr><tr><td> anti-insan CD19-PE </td><td> BECKMAN COULTER </td><td> A07769 </td><td></td></tr><tr><td> anti-insan CD45-ECD </td><td> BECKMAN COULTER </td><td> A07784 </td><td></td></tr><tr><td> anti-insan CD56-PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07789 </td><td></td></tr><tr><td> anti-insan TCRαβ-PE </td><td> BECKMAN COULTER </td><td> A39499 </td><td></td></tr><tr><td> TCR Vγ9 anti-insan-FITC </td><td> BECKMAN COULTER </td><td> IM1463 </td><td></td></tr><tr><td> anti-insan CD27-PE/Cy5 </td><td> BECKMAN COULTER </td><td> 6607107 </td><td></td></tr><tr><td> anti-insan CD45RA-ECD </td><td> BECKMAN COULTER </td><td> IM2711 </td><td></td></tr><tr><td> anti-insan CD69-PE </td><td> BD </td><td> 555531 </td><td></td></tr><tr><td> anti-insan NKG2D-PE </td><td> BECKMAN COULTER </td><td> A08934 </td><td></td></tr><tr><td> Anti-humal IFNγ-PE </td><td> BECKMAN COULTER </td><td> IM2717U </td><td></td></tr><tr><td> Fare IgG1 izotip kontrol PE </td><td> BECKMAN COULTER </td><td> A07796 </td><td></td></tr><tr><td> Mouse IgG1 izotip kontrol ECD veya PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07797 A07798 </td><td></td></tr></tbody></table>

expansion of human peripheral blood  &gamma;&delta; t cells using zoledronate

İnsan γδ T hücreleri tanımak ve stres kaynaklı antijenleri geniş bir yelpazede cevap, böylece doğuştan gelen geniş bir anti-tümör ve anti-enfektif aktivite gelişmekte olan 1, periferik kan γδ T hücrelerinin çoğunluğu Vγ9Vδ2 T hücre reseptör var . Bu hücreler, büyük doku uygunluk kompleksi-bağımsız bir şekilde antijeni tanıması ve güçlü sitolitik ve Th1 gibi efektör fonksiyonları gelişir. 1 Bu nedenle, γδ T hücrelerinin, kanser immünoterapi için cazip aday efektör hücreleri vardır. Vγ9Vδ2 T hücreleri gibi phosphoantigens cevap (E)-4-hidroksi-3-metil-ama-2-enyl pirofosfat (HMBPP), izoprenoid biyosentezi ile bakteriler sentezlenir; üretilen 2 ve izopentenil pirofosfat (IPP), mevalonata yolu 3 fizyolojik durumu ile ökaryot hücreler, IPP nontransformed hücre üretimi γδ T hücrelerinin aktivasyonu için yeterli değildir. Düzensizliği, tümör hücrelerinde mevalonata yolun IPP birikimi ve γδ T hücrelerinin aktivasyonu yol açar. 3 aminobisphosphonates (örneğin, pamidronat veya zoledronate) farnesil pirofosfat sentaz (SOP), mevalonata yolu IPP mansabında hareket eden enzim, hücre içi düzeylerini inhibe Çünkü γδ T hücreleri tanıma IPP ve sensitibity terapötik aminobisphosphonates artabilir. IPP birikimi nontransfomred hücreleri aminobisphosphonates ile γδ T hücreleri aktive ederek kanser için bize izin immünoterapi aminobisphosphonates farmakolojik ilgili bir konsantrasyon ile tümör hücrelerinin daha az verimlidir. PBMC aminobisphosphonates ile tedavi edildiklerinde 4 İlginçtir, IPP çünkü verimli, monositler birikir bu hücreler tarafından ilaç alımı. IPP antijen-sunan hücreler haline ve periferik kan Vγ9Vδ2 T hücreleri uyarmak birikir 5 Monositler 6 bu mekanizmalar dayanarak, biz γδ T hücre kültürleri zoledronate ve interlökin kullanarak büyük ölçekli genişlemesi için bir teknik geliştirdi -2 (IL-2). sentetik phosphoantigens bromohydrin pirofosfat (BrHPP) 8 ya da 2-metil-3-butenyl-1-pirofosfat (2M3B1PP) 7 γδ T hücrelerinin genişlemesi için diğer yöntemler kullanmaktadır. 9 Tüm bu yöntemlerin eski izin evlatlık immünoterapi kullanmak için γδ T hücrelerinin çok sayıda in vivo genişlemesi. Ancak, zoledronate sadece bir FDA onaylı piyasada bulunan reaktif. CD45RA - - Zoledronate-genişletilmiş γδ T hücreleri CD27 ekran efektör bellek fenotipi ve fonksiyonu IFN-γ üretimi tahlil 7 değerlendirilebilir.

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  Î³Î´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

Γδ T hücreleri periferik kan mononükleer hücreleri (hücre) genişletmek için bir yöntem açıklanmıştır. Hücre-kökenli γδ T hücreleri uyarılır ve zoledronate ve interlökin-2 (IL-2) ile genişletilir. Γδ T hücrelerinin büyük ölçekli genişlemesi kanser otolog hücresel immünoterapi uygulanabilir.

Zoledronate İnsan Periferik Kan Genişleme γδ T Hücreler

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

expansion-of-human-peripheral-blood-t-cells-using-zoledronate

Research

JoVE Journal

Immunology and Infection

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

26.3K Görüntüleme.  University of Tokyo Hospital. Γδ T hücreleri periferik kan mononükleer hücreleri (hücre) genişletmek için bir yöntem açıklanmıştır. Hücre-kökenli γδ T hücreleri uyarılır ve zoledronate ve interlökin-2 (IL-2) ile genişletilir. Γδ T hücrelerinin büyük ölçekli genişlemesi kanser otolog hücresel immünoterapi uygulanabilir.

Video: Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Genişleme, Saflaştırılması, ve İnsan Periferik Kan NK Hücrelerinin Fonksiyonel Değerlendirme

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

İmmünoloji ve Enfeksiyon

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Klinik Uygulama<em&gt; Sleeping Beauty</emGenetiği Periferik ve Göbek Kordon Kanı alınan T hücreleri Değiştir Sunan Hücreler&gt; ve Yapay Antijen

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

İnsan Endoservikal Gamma Delta T Hücreleri İzolasyon ve Akım Sitometrisi Analizi

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran fare ve insan birincil NK hücreleri Lentiviral iletim verimliliğini artırır

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Madstorm kullanarak T hücrelerinin çok yönlü, süper çözünürlüklü görüntüsü

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Periferik kan ve lenf dokusu birincil insan T hücre sinaptik arabirimi değerlendirilmesi

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Flotasyon Bazlı T Hücresi İzolasyonu, Aktivasyonu ve Mikrokabarcıklar Kullanılarak İnsan Periferik Kan Mononükleer Hücre Örneklerinden Genişleme

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Fonksiyonel Osteoklastların İnsan Periferik Kanı CD14+ Monositlerinden Farklılaşması

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Mitokondriyal Biyoenerjetik Analizi için PBMC Toplama ve Kriyo Depolama

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Yazar Spotlight: Kriyoprezervasyon Sonrası Periferik Kan Mononükleer Hücrelerinde Biyoenerjetik Parametrelerin Korunması 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

İnsan Periferik Kan Mononükleer Hücrelerinin Kriyoprezervasyonu ve Biyoenerjetik Değerlendirmesi

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...