Bu çalışmanın yanı sıra, National Institutes of Health DK074656 ve AI56374 (CEM) hibe Juvenil Diyabet Araştırma Vakfı tarafından desteklenmiştir.

1. Hücrelerin hazırlanması <ol><li> Hedef hücre kültürü: Kültür fare beta hücre hatları, NIT-1 ve NIT-4,% 10 FBS,% 0.02 BSA, Non-esansiyel amino asit, 15mm Hepes, 16.5mm ile desteklenmiş DMEM önceden açıklanan 1,2 oldu Glocose ve Penisilin / Streptomisin. 5x10 4 hücre düz bir alt 96-kuyu kültür plaka 51 Cr etiketleme, tohum hücreleri / 200 ul kültür ortamı iyi. Mikroskopi deneyler, 500 ul kültür ortamı oda başına 5x10 4 8-iyi Lab-Tak II odacıklı coverglass tohum hücreleri ve sitotoksisite deneyleri kullanarak 48 saat önce büyümek için izin. </li><li> Kontrol T hücre hattı kültürü: (ATCC, CD3 + insan lenfosit hücre hattı) negatif kontrol olarak Jurkat hücre hattı kullanın. RPMI1640 Kültür Jurkat hücrelerin% 10 FBS ile desteklenmiş, 2 mM L-glutamin, 1.5 g / L sodyum bikarbonat, 10 mM Hepes ve 1.0 mM sodyum piruvat. </li></ol> 2. Efektör otoreaktif CD8 + T hücrelerinin toplanması ve aktivasyonu NOD.Cg Rag1 tm1Mom Tg (TcraAI4) 1Dvs/DvsJ [NOD-AI4a] ve NOD.Cg Rag1 tm1Mom Tg (TcrbAI4) 1Dvs/DvsJ [NOD-AI4b] Jackson Laboratuvarı (Bar Harbor, ME) satın alınan ve fare tesiste yetiştirilen. NOD-AI4a çiftleşmelerin F1 hibrit döl NOD-AI4b [NOD-AI4a / b] 3-5 yaş hafta diyabet gelişecektir. Tüm fareler spesifik patojen free tesisleri içinde yer alır ve ilgili kurum Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. <ol><li> CO 2 kullanarak 3-4 hafta eski NOD-AI4a / b fareler Euthanize. </li><li> Farelerden alınan dalak toplayın. Ötenazi fareler etanol sprey ile hazırlanan ve cerrahi kaputun altında açıldı. Dalak çıkarılır ve hemen buz HBSS tampon konur. </li><li> 7 ml&#39;lik cam homojenizatör kullanarak dalak hücrelerinin toplayın. İki üç dalak her 5 ml soğuk HBSS koymak ve 7 ml&#39;lik cam homojenizatör ile homojenize edilir. Homojenat 4 1200rpm 5 dakika boyunca toplanan ve santrifüj ° C Sorvall RT7 + santrifüj kullanarak. Hücre pelletleri toplanır. </li><li> Hipotonik çözüm tedavisi kullanarak kırmızı kan hücrelerinin çıkarın. Peletler, 5 dakika buz üzerinde çözüm / dalak 5 ml hipotonik ile tedavi edilir, daha sonra eşit miktarda HBSS ekleyerek nötralize. Yukarıdaki gibi, santrifüj hücreleri toplayın ve 50 ml HBSS tekrar süspansiyon haline getirin. Askıya alınan hücrelerin hücre süzgecinden Pass ve TrypanBlue boyama ile hemasitometre kullanarak saymak. </li><li> Hücreleri aktive. 5x10 6 / ml ve etkinleştirmek RPMI1640 0.1 mcM AI4 mimotope (amino asit dizilimi YFIENYLEL) ve 25 U / ml, IL-2 ile 3 günlük kültür kullanarak yeniden süspanse hücrelerin% 10 FBS ile desteklenmiş, 2 mM L-glutamin, 1.5 g / L sodyum bikarbonat, 10 mM HEPES ve 1.0 mM sodyum piruvat. </li></ol> 3. 51 Cr serbest tahlil KML incelemek için <ol><li> Etiket hedef hücreleri: 1 ile 3 saat μCi / iyi ve kültür hücreleri hedef ve kültür ortamı ile 3 kere yıkayın 51 Cr ekleyin. </li><li> Son hacmi, her bir kuyunun eklendi böylece adım 2.5 &#39;te belirtilen RPMI 1640 orta efektör hücreleri Askıya Alma 200 ul. </li><li> Etiketli hedef hücreleri son yıkamadan sonra, oranları: (T E) hedef, istenilen efektör hedef hücre kültürleri efektör hücreleri aktive eklemek. 0.4:1, 1:1, 2:1, 5:1, 10:01, 20:01 ve 50:1 T oranları: Biz genellikle E kullanabilirsiniz. </li><li> Kendiliğinden bir parçalama kontrolü gibi hareket etmek, hedef hücrelerin 6 kuyu taze değiştirilmiş RPMI 1640 orta (2.5 adım) ekleyin. </li><li> 16 saat boyunca ko-kültür efektör ve hedef hücreleri. </li><li> 6x50 mm Kireç Cam tüpler supernatant toplayın </li><li> 100μl% 2 SDS ekleme ve hafifçe birkaç kez pipetleme hücreleri Lyse. </li><li> 6x50 mm Kireç Cam tüpler başka bir dizi hücre lizat toplayın. </li><li> Temiz H 2 O kuyuların bir kez yıkayın ve hücre lizat tüplerine yıkama ekleyin. </li><li> Bir gama sayacı kullanarak süpernatantlar ve hücre lizatları güvenin. </li><li> Özel lizis aşağıdaki gibi hesaplayın: <img src="files/ftp_upload/2724/2724eq1.jpg" alt="Denklem 1" /></li></ol> 4. Canlı hücre görüntüleme için hücrelerin boyanması En sık kullanılan mitokondriyal membran potansiyeli boyalar arasında TMRM en mitokondriyal toksisite sergiler. Bu nedenle 3, biz bu canlı hücre görüntüleme çalışmaları için TMRM seçin. <ol><li> DMSO ve mağaza -20 ° C&#39;de 40 mM stok TMRM çözüm hazırlayın [25 nM] NIT-1 hücre kültürü 1 için tüm takviyeleri ile fenol kırmızısı DMEM taze çalışma çözüm olun. </li><li> 37 ° 30 dakika TMRM 25 nM hedef NIT-1 hücreleri leke ° C </li><li> Boya 4 denge dağılımı korumak için TMRM 5 nM içeren taze medya ile medya değiştirin. </li><li> Hematocytometer kullanarak aktive efektör AI4 T hücreleri güvenin. </li><li> 1 ul / ml Picogreen için aktif efektör AI4 T hücreleri LekeOda sıcaklığında 5 dakika ve fenol kırmızısı içermeyen kültür ortamı 3 kez ile yıkayın. </li></ol> 5. Canlı hücre görüntüleme kullanılarak değerlendirilmesi T lenfosit aracılı beta hücre ölümü <ol><li> Odacıklı coverglass Zeiss LSM 510 konfokal mikroskop aşamasında lekeli NIT hücreleri ile monte edin. Dremmel ve gaz odacıklı cam etheline oksit ile sterilize sekmesini çıkarın. Aşamada, sıcaklık 37 ° ° C ve% 5 CO 2 nem ile. Olan bir canlı hücre görüntüleme odası ile donatılmıştır . </li><li> Aktive ekleyin ve farklı E belirlenen odalarına lekeli T hücreleri: T oranları. </li><li> Lekeli Jurkat hücrelerinin aynı sayıda kontrol odalarına eklendi. </li></ol> 6. Canlı hücre görüntüler Edinme Mikroskop, bize tekrar tekrar deney süresi boyunca otomatik olarak aynı ya da farklı odalarına farklı yerlerde görüntüler elde etmek için izin motorlu bir sahne ile donatılmıştır. <ol><li> 63x yağ objektif lens kullanarak, konumu başına 300 kare olmak üzere toplam her 3 dakikada bir aralıkta görüntüleri alır. </li><li> Emisyon 565-619 nm eksitasyon dalga uzunluğu 543 nm ve filtre seti kullanarak TMRM boyama Algılama </li><li> Algılama bir uyarım 488 nm ve emisyon 500-630 nm için bir filtre kullanılarak Picogreen boyama. Video Zeiss LSM yazılımı kullanılarak oluşturuldu. </li></ol> 7. Yayın için video dönüştürme <ol><li> Zeiss LSM yazılımı kullanarak, sıkıştırılmamış bir AVI formatında film dosyası oluşturabilirsiniz. </li><li> Video yayınlanan biçime dönüştürmek için, video, yazılım paketi Fiji (içine ithal <a href="http://pacific.mpi-cbg.de/wiki/index.php/Fiji">http://pacific.mpi-cbg.de/wiki/index.php/Fiji</a> ), bir ImageJ ( <a href="http://rsb.info.nih.gov/ij/">http://rsb.info. nih.gov / ij /</a> lokusların (Bioformats eklentisi kullanarak) dağıtım, <a href="http://www.loci.wisc.edu/software/bio-formats">http://www.loci.wisc.edu/software/bio-formats</a> ). </li><li> Yeşil ve kırmızı sinyaller ve kare hızı, animasyon seçenekler menüsünü kullanarak, 2 kare / saniye olarak ayarlandı dengesi, parlaklık ve kontrast ayarlarını ayarlayın. </li><li> Sıkıştırılmamış bir AVI dosyası olarak 512 x 512 piksel ve ihracat için video kırpın. </li><li> Yazılım Quicktime 7 (Apple bilgisayar, Cupertino, CA) bu AVI dosyasını açın ve sıkıştırmak ve açık video dosyası formatı H264/MPEG-4 1200 kbs / ikinci ihracat. </li></ol> 8. Temsilcisi Sonuçlar: <ol><li> 51 Cr KML testinin bir temsilci sonucudur. Hedef oranı (X ekseni) artar: Şekil 1, yüzde belirli lizis (Y ekseni) Efektör artar bir KML testinin temsili bir sonuç göstermektedir. Hedef hücrelerin immünyetmezligi NOD izole birincil adacıklar Rag1-/ - fareler ve 51 Cr etiketli. NOD.AI4α / β transgenik fareler izole dalak ve yukarıdaki 2. adımda açıklandığı gibi aktive edilir. Hedefler ve etkileyiciler 16 saat için ortak kültüre edildi. Yüzde özel lizis yukarıda adım 3.11 &#39;de açıklandığı gibi hesaplanır. </li><li> T hücre-aracılı beta hücre mitokondrial membran potansiyeli dağılımı iki temsilci (pozitif ve negatif) sonuçları sunulmaktadır. Hücreler lekeli ve reaksiyonlar adımları 4, 5 ve 6 yukarıda anlatıldığı gibi kayıt altına alınır, video dosyaları, 7. adımda olarak oluşturulur. Video 1: fare beta hücre hattı NIT-1 mitokondrial membran potansiyeli boya TMRM (Kırmızı) boyandı. T oranı 50:1: Aktive, otoreaktif CD8 + T hücre AI4 (Picogreen Vitray yeşil) E bu NIT-1 hücreleri ile ortak kültüre edildi. NIT-1 mitokondrial membran potansiyeli 400 dakika süresince yavaş yavaş dağıldı, ancak sadece AI4 T hücreleri ile etkileşim içinde olan kümeler. Video 2: Bir kontrol deneyi olarak, NIT-1 hücreleri TMRM ve E Picogreen lekeli Jurkat hücreleri ile ko-kültür ile boyandı: T oranı 50:1. Jurkat hücreleri MHC uyumsuz bir insan T hücre hattı. NIT-1 hücreleri co-inkübasyon süresi 400 dakika tüm süresi boyunca mitokondrial membran potansiyeli sürdürdü. Jurkat hücreleri NIT-1 kümeleri ile etkileşim yoktu ve bu nedenle öldürülmesi neden yok. </li></ol><img src="files/ftp_upload/2724/2724fig1.jpg" alt="Şekil 1" /> Şekil 1 KML Temsilcisi sonucu immünyetmezligi NOD.Rag1 / izole hedef hücreleri birincil adacıklar olarak kullanarak fare ve dalak NOD.AI4α / β transgenik fareler. Hedefler ve etkileyiciler 16 saat için ortak kültüre edildi. Yüzde özel lizis Efektör olarak artar: Hedef oranını yükseltir. Video 1 Mouse beta hücre hattı NIT-1 mitokondriyal membran potansiyeli boya TMRM (Kırmızı) boyandı. Aktive otoreaktif CD8 + T hücre AI4 (Vitray Picogreen ile yeşil) bu NIT-1 E hücreleri ile ortak kültüre edildi: 50:1 T oranı. NIT-1 mitokondrial membran potansiyeli dissipaTed kademeli olarak 400 dakikalık süre içinde, ama sadece yeşil AI4 T hücreleri ile etkileşim içinde olan kümeler. <a href="http://www.jove.com/files/ftp_upload/2724/video.mov">videosunu izlemek için tıklayın</a> . Video 2 Video 1 ve ko-kültür Picogreen lekeli insan T hücre hattı Jurkat açıklandığı gibi NIT-1 hücreleri aynı boyandı. NIT-1 hücrelerinde mitokondriyal membran potansiyeli 400 dakika süre düşünülmüş koruyabilmiştir. NIT-1 kümeleri Jurkat hücreleri etkileşim yoktu ve bu nedenle öldürülmesi neden yok. <a href="http://www.jove.com/files/ftp_upload/2724/Video2.mp4">videosunu izlemek için buraya tıklayın</a> .

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Üniversitesi Florida Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

Sitotoksik T hücre aracılı beta hücre ölümü T1D 5 ana patofizyolojisi. 51 Cr sürümü kullanarak KML testleri bize efektör-hedef yanıt 6 derecesini incelemek için izin verir. Ancak, ayrıntılı bir süreç ve T hücre-aracılı beta hücre ölümü yolları hala tam olarak anlaşılmış değildir. Mitokondri, beta hücre fonksiyonu ve ölüm 7 için kritik olduğundan, görsel KML sırasında mitokondrial değişiklikler üzerine odaklanmıştır . Mitokondrial za...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Cr-51 </td><td> Perkin Elmer </td><td> NEZ030500IMC </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-60 </td><td></td></tr><tr><td> Tetramethyl rodamin Metil Ester (TMRM) </td><td> Invitrogen </td><td> T668 </td><td></td></tr><tr><td> Picogreen </td><td> Invitrogen </td><td> P7581 </td><td></td></tr><tr><td> AI4 mimotope </td><td> mimotopes.com </td><td> amino asit dizilimi: YFIENYLEL </td><td></td></tr><tr><td> IL-2 </td><td> Ar-Ge </td><td> 485-MI </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> FBS </td><td> HyClone </td><td> SH30071.03 </td><td></td></tr><tr><td> Sığır Serum Albumin </td><td> Sigma </td><td> A8412 </td><td></td></tr><tr><td> Hepes </td><td> Cellgro </td><td> 25-060-Cl </td><td></td></tr><tr><td> MEM Non-Essential Amino Asitler </td><td> Gibco </td><td> 11140 </td><td></td></tr><tr><td> Penisilin / Streptomisin </td><td> İkizler burcu </td><td> 400-109 </td><td></td></tr><tr><td> Fenol kırmızı ücretsiz DMEM </td><td> Sigma </td><td> D5303 </td><td></td></tr><tr><td> CO2 İnkübatör </td><td> Thermo Fisher </td><td> HeraCell 150i </td><td> Hücre kültürü için steril Kept </td></tr><tr><td> Biyolojik güvenlik kabini </td><td> Thermo Fisher </td><td> Forma 1440 </td><td></td></tr><tr><td> Tek kullanımlık kültür tüpleri, 6x50 mm Kireç Cam </td><td> Balıkçı </td><td> 14-958-A </td><td></td></tr><tr><td> Gama Sayıcı </td><td> Perkin Elmer </td><td> Wizard 1470 Otomatik Gama Sayıcı </td><td></td></tr><tr><td> Konfokal mikroskop </td><td> Zeiss </td><td> EKK 510 Meta Konfokal </td><td> Motorlu sahne ve canlı hücre odasına </td></tr><tr><td> Pipet (1ml, 200μl, 20μl, 10μl, 2μl) </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Borular (Sarı) </td><td> Balıkçı </td><td> 50819772 </td><td></td></tr><tr><td> Santrifüj </td><td> Sorvall Legend RT + </td><td> 75004377 </td><td></td></tr><tr><td> Hemasitometre </td><td> Fisher Scientific </td><td> 267110 </td><td></td></tr><tr><td> Dik Mikroskop </td><td> Zeiss </td><td> Axioskop40 </td><td></td></tr><tr><td> NOD.Cg Rag1 tm1Mom Tg (TcraAI4) 1Dvs/DvsJ Fare </td><td> Jackson Laboratuvarı </td><td> 004347 </td><td></td></tr><tr><td> NOD.Cg Rag1 tm1Mom Tg (TcrbAI4) 1Dvs/DvsJ Fare </td><td> Jackson Laboratuvarı </td><td> 004348 </td><td></td></tr><tr><td> NOD-AI4α / β F1 fareler </td><td> N / A </td><td> N / A </td><td> UF hayvan tesisi yetiştirilen </td></tr></tbody></table>

methods to assess beta cell death mediated by cytotoxic t lymphocytes

Tip 1 diyabet (T1D) T hücre aracılı otoimmün hastalık. Patogenezinde sırasında, hastaların insülin üretimi kayıp olarak T hücreleri tarafından pankreas beta hücrelerinin yıkımını muhtemelen bu sonuç, giderek daha insulinopenic hale gelir. T1D gelişimi sırasında beta hücre ölüm mekanizmaları anlamak, bu hastalık için etkili bir çare üretmek için anlayışlar sağlayacaktır. Hücre aracılı lenfositotoksisite (KML) testlerin tarihsel hedef hücreleri etiket radyonüklid Krom 51 (51 Cr) kullanılır. Bu hedefler daha sonra efektör hücreleri maruz kalan ve 51 Cr hedef hücrelerden serbest bırakılması lenfosit aracılı hücre ölümü bir göstergesi olarak okunur . 51 Cr azalmış serbest hücre ölümü sonucu inhibitörleri. Efektör hücreler olarak, CD8 aktive otoreaktif klonal nüfus + AI4 T hücre reseptör (TCR) alfa ve beta zincirleri için bir fare stok transgenik izole Sitotoksik T lenfositler (CTL) kullanılır. Aktif AI4 T hücreleri 16 saat boyunca 51 Cr etiketli hedef NIT hücreleri birlikte kültür, serbest bırakılması 51 Cr belirli lizis hesaplamak için kaydedildi Mitokondri, enerji üretimi, sinyal transdüksiyon düzenlenmesi ve apoptosis gibi birçok önemli fizyolojik olaylar, katılırlar. T1D gelişimi sırasında beta hücre mitokondrial fonksiyonel değişiklikler çalışma, bir araştırma roman alanıdır. Mitokondriyal membran potansiyeli boya tetramethyl rodamin Metil Ester (TMRM) ve konfokal mikroskobik canlı hücre görüntüleme kullanarak, beta hücre hattı NIT-1 mitokondrial membran potansiyeli zaman içinde izlenmektedir. Görüntüleme çalışmaları için, efektör AI4 T hücreleri floresan nükleer boyanma boya Picogreen etiketli. NIT-1 ve T hücreleri odacıklı coverglass ortak kültür ve canlı bir hücre odası ile donatılmış mikroskop aşamasında üzerine monte edilmiş, 37 kontrol edildi ° C,% 5 CO 2 ve nemlendirilmelidir. Bu deneyler sırasında görüntüleri her küme her 3 dakikada bir 400 dakika alındı. 400 dakika boyunca, biz AI4 T hücreleri bağlı NIT-1 hücre kümeleri mitokondrial membran potansiyeli dağılımı görülmektedir. NIT-1 hücreleri MHC ile birlikte kültüre edildi eşzamanlı kontrol deneyde yanlış uyumlu insan lenfosit Jurkat hücreleri, mitokondriyal membran potansiyeli bozulmadan kalabilmiş. Bu teknik, sitotoksik lenfosit, sitokinler, veya diğer sitotoksik reaktifler saldırı altında hücreleri mitokondrial membran potansiyeli gerçek zamanlı değişiklikleri gözlemlemek için kullanılabilir.

Watch this Scientific Journal Video about Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes at JoVE.com

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Hücre-aracılı lenfositotoksisite (KML) deneyleri, in vitro otoreaktif yanıtları ve hücre ölümü çalışma mekanizmaları test etmek için kullanılabilir. Ancak, canlı hücre floresan boyalar, tipi ve hücre ölümü gibi kullanılan yollar kinetiği konfokal mikroskopik görüntüleme teknikleri kullanılarak daha ayrıntılı olarak incelenebilir.

Sitotoksik T Lenfositler Beta Hücre Ölümü aracılığı ile değerlendirin yöntemleri

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

methods-to-assess-beta-cell-death-mediated-by-cytotoxic-t-lymphocytes

Research

JoVE Journal

Immunology and Infection

13.3K Görüntüleme.  University of Florida. Hücre-aracılı lenfositotoksisite (KML) deneyleri, in vitro otoreaktif yanıtları ve hücre ölümü çalışma mekanizmaları test etmek için kullanılabilir. Ancak, canlı hücre floresan boyalar, tipi ve hücre ölümü gibi kullanılan yollar kinetiği konfokal mikroskopik görüntüleme teknikleri kullanılarak daha ayrıntılı olarak incelenebilir.

Video: Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Kordon Kanı adlı Sitotoksik T lenfositler genişletilmesi olduğunu Hedef Sitomegalovirüs, Epstein-Barr Virus ve Adenovirüs

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

İmmünoloji ve Enfeksiyon

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

XCELLigence Sistemi Cr51 Yayın Assay Karşılaştırılması tarafından İnsan Her2neu Özgül CD8 T hücrelerinin Optimal Sitotoksik Aktivite Belirlenmesi

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

T Lenfositler doğru İsteyerek Pluripotent Kök Hücrelerinin Yönetmen Farklılaşma

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Radyal Hareket ve retroviral replike olan bir vektör transduse, Yapışmayan Alloresponsive T lenfositlerinin sitotoksik işlev

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Flüoresan Korelasyon Spektroskopisi Ölçülen İlköğretim Lenfositler Plazma Membranlarda moleküler difüzyon

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Bağışıklık ilaçlarının tanımlanmasında T lenfositlerinde mitojen kaynaklı blastojenez hızlı bir şekilde belirlenmesini

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Grip Hemagglutinin özel antikorlar tarafından indüklenen Fc-aracılı efektör fonksiyonları değerlendirmek için bir yöntem

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

Terminal transferaz-aracılı dUTP kullanarak Nick uç-etiketleme (tünel) ve Caspase 3/7 deneyleri için ölçü Epidermal hücre ölümünde kurbağalar Chytridiomycosis ile

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...