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.

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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  ÃŽÂ³ÃŽÂ´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  &#947;&#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

26.5K 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  γδ 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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

İlköğretim İnsan naif ve Bellek T Hücreleri gelen İsteyerek Düzenleyici T Hücreleri Üretimi

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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

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

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

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

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

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

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 ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

İnsan Periferik Kan immünfenotipik Analizi Antikor Kokteyller Hazırlama A Yarı-otomatik Yaklaşım

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

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.