وأيد هذا العمل من قبل L. دان دنكان منحة بحثية تعاونية (CMB ونقابة الصحفيين المصرية)، المعهد الوطني للقلب والرئة، والدم المعهد (US4HL081007)، وسرطان الدم وسرطان الغدد الليمفاوية جمعية جائزة الباحث العلمي البحوث السريرية (CMB)، والمعهد الوطني للسرطان (RO1 CA06150816؛ نقابة الصحفيين المصرية).

1. وحيدات النوى خلية عزل (يوم 0) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إدراج محول ارتفاع الأنبوب الإناث في ميناء مخرج الكسر 20٪ من الحبل وحدة الدم، وإزالة إرفاق حقنة الدم. نقل الدم إذابة إلى 20 مل من RPMI تحسنت في 50 مل أنبوب الطرد المركزي. شطف كيس الدم الحبل مع 5 مل من RPMI ونقل إلى أنبوب الطرد المركزي نفسه. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الخلايا لمدة 10 دقائق في ز × 400. نضح طاف. </li><li style=";text-align:right;direction:rtl"> إعادة تعليق الخلايا في 20 مل من RPMI الدافئة. خلايا طبقة على 15 مل من Lymphoprep في 50 مل أنبوب الطرد المركزي. أجهزة الطرد المركزي 40 دقيقة @ 400 × ز. </li><li style=";text-align:right;direction:rtl"> حصاد واجهة تحتوي على الخلايا وحيدات النوى، وأغسلها في 20 مل من RPMI. أجهزة الطرد المركزي لمدة 10 دقائق @ 450 × ز. </li><li style=";text-align:right;direction:rtl"> نضح طاف ويغسل في 20 مل من RPMI. عد الخلايا وتدور لمدة 5 دقائق @ 400 × ز. </li><li style=";text-align:right;direction:rtl"> نضح طاف الخلايا و resuspend في 5 × 10 6 خلايا وحيدات النوى / مل من وسائل الإعلام Cellgenix DC (المصل مجانا). 1 مل لإزالة EBV-LCL جيل وضعت في 15 مل مntrifuge الأنبوب. </li></ol> 2. شجيري الجيل الخليوي (بدءا من اليوم 0) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لوحة 2 مل من الخلايا (هي بالفعل في 5 × 10 6 خلية / مل من وسائل الإعلام DC) لكل بئر في لوحة 6 أيضا. ترك في حاضنة لمدة 1-2 ساعات. </li><li style=";text-align:right;direction:rtl"> بعد 1-2 ساعات، يغسل خلايا غير ملتصقة عن طريق الغسيل 3-4 مرات مع برنامج تلفزيوني. جمع الخلايا غير ملتصقة والتجميد. إضافة 2 مل / جيد من وسائل الإعلام DC تحتوي على 1000 U / مل من IL-4 و 800 U / مل GM-CSF. </li><li style=";text-align:right;direction:rtl"> 3-4 أيام بعد بدء DC، تجديد IL-4-CSF وجنرال موتورز عن طريق إضافة 100 ميكرولتر / جيد من وسائل الإعلام التي تحتوي على تركيز النهائي من 1000U/mL IL-4 و 800 U / مل GM-CSF. </li><li style=";text-align:right;direction:rtl"> في يوم 5-6، حصاد DC عن طريق كشط البئر مع ماصة نقل. عد الخلايا الكبيرة وأجهزة الطرد المركزي لمدة 5 دقائق @ 400 × ز. الرشفة وسائل الإعلام وDC إعادة تعليق على 2X10 6 خلايا / مل من وسائل الإعلام DC. إضافة 0.5 مل / بئر لوحة 24 أيضا. </li><li style=";text-align:right;direction:rtl"> تنبيغ وDC باستخدام ناقلات Ad5f35pp65 في خلية في وزارة الداخلية من 10 وحدات المعدية. إضافة vectoR لكل بئر في لوحة 24 أيضا. احتضان خلايا مقابل 1.5 ساعة. بعد 1.5 ساعة، إضافة 1.5 مل / DC جيدا وسائل الإعلام التي تحتوي على: 1000 U / مل IL-4، 800 U / مل GM-CSF، 10 نانوغرام / مل TNF-ألفا، 1 ميكروغرام / مل PGE-1، 100 نانوغرام / مل IL-6، و 10 نانوغرام / مل IL-1beta. هذه السيتوكينات انضاج البلدان النامية. </li></ol> 3. جيل من EBV-LCL (بدءا من اليوم 0) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في أنبوب يحتوي على 5 × 10 6 خلايا وحيدات النوى. أضف 200 ميكرولتر من طاف تتركز B95.8 EBV و 1.8 مل من وسائل الإعلام كاملة (+ 10٪ RPMI FBS + L-2mm ​​والجلوتامين) التي تحتوي على السيكلوسبورين (1 ميكروغرام / مل). </li><li style=";text-align:right;direction:rtl"> قسامة 100 ميكرولتر من الخلايا في 10 آبار من لوحة 96-جيدا و 200 ميكرولتر إلى 5 آبار. إضافة 100 ميكرولتر من وسائل الإعلام كاملة + السيكلوسبورين إلى الآبار التي تحتوي على فقط 100 ميكرولتر من الخلايا. ملء الآبار المتبقية مع الماء المعقم. </li><li style=";text-align:right;direction:rtl"> تغذية الأسبوعية LCL والتوسع من لوحة 96-جيدا للوحة جيدا بعد 24 أسابيع 2 ~. بعد أسبوع آخر، ونقل إلى قارورة T25 ونقل في الأسبوع اللاحققارورة T75. في هذه المرحلة فمن المستحسن أن تقوم بتجميد مأخوذة من LCL. يستغرق عادة 1 شهر لتوليد عدد كاف من LCL. </li></ol> 4. بدء CTL - 7 أيام بعد بدء البلدان النامية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> البلدان النامية الحصاد وأشرق في 30 غراي. 4 × غسل والعد. إعادة تعليق DC في وسائل الإعلام التي تحتوي على مصل الخلية T الإنسان (45٪ RPMI، 45٪ CLICK، ومصل الإنسان 10٪، 2mm والجلوتامين L-) @ 1 × 10 5 DCS / مل. </li><li style=";text-align:right;direction:rtl"> ذوبان الجليد غير ملتصقة الخلايا من الخطوة 2.2. غسل الخلايا، والعد. إعادة تعليق في 2 × 10 6 غير ملتصقة خلية / مل في وسائل الإعلام التي تحتوي على مصل الخلية T الإنسان. إضافة 10 نانوغرام / مل IL-7 12-IL و، و 5 نانوغرام / مل IL-15. إضافة 1 مل من الخلايا لكل بئر في لوحة 24 أيضا. إضافة 1 مل من DC لكل بئر. ملء الآبار فارغة من 24 لوحة جيدا مع الماء المعقم. </li><li style=";text-align:right;direction:rtl"> 7 أيام بعد بدء خلية T، تغذية و / أو تقسيم خلايا مع وسائل الإعلام التي تحتوي على مصل الخلية T الإنسان. </li><li style=";text-align:right;direction:rtl"> 9-12 أيام بعد بدء خلية T، وتجميد الخلايا T حتى LCL جاهزة. </li><li style=";text-align:right;direction:rtl"> ONCE LCL توسعت وpassaged في قوارير T75، وتنبيغ LCL بواسطة الطرد المركزي لمدة 5 دقائق @ 400 × ز. إضافة إلى ناقلات بيليه الخلية في وزارة الداخلية على 100 وحدة المعدية في الخلية. احتضان لمدة 1.5 ساعة. بعد 1.5 ساعة، resuspend في وسائل الإعلام الكامل في 5 × 10 5 مل / LCL وإضافة 2 مل لكل بئر في لوحة 24 أيضا 12 </li><li style=";text-align:right;direction:rtl"> بعد 2 يوم، حصاد LCL وأشرق في 40 غراي. 4 × غسل والعد. إعادة تعليق على LCL في وسائل الإعلام التي تحتوي على مصل الخلية T الإنسان عند 2.5 X 5 10 خلايا / مل. إضافة 1 مل من LCL لكل بئر من لوحة 24 أيضا. أيضا، إضافة 5 × 10 6 LCL إلى جهاز الثقافة Grex40 وكذلك 5 نانوغرام / مل IL 15-13 </li><li style=";text-align:right;direction:rtl"> حصاد الخلايا T. أجهزة الطرد المركزي لمدة 5 دقائق @ 400 XG، نضح طاف، و resuspend في وسائل الإعلام التي تحتوي على مصل الخلية T الإنسان في 1 × 10 6 خلايا T / مل. إضافة 5 نانوغرام / مل IL-15 وإضافة 1 مل / جيد من الخلايا T (مجموعه 1 × 10 خلايا T 6) إلى LCL التي هي بالفعل في لوحة 24 أيضا. في Grex40، وجلبالحجم الكلي للسائل الإعلام تصل إلى 30 مل. </li><li style=";text-align:right;direction:rtl"> في يوم 3-4 التحفيز آخر، إذا كانت الخلايا متموجة أدى ذلك إلى انقسام لهم (في لوحة) 1:1 وسائل الإعلام إضافة جديدة تحتوي على 50 U / مل IL-2. إذا لم تكن متكدسة ثم نضح ببساطة ½ وسائل الإعلام واستبدالها مع وسائل الإعلام التي تحتوي على 50 U / مل IL-2. في Grex40، نضح نصف وسائل الإعلام، استبدال مع وسائل الإعلام الجديدة، وإضافة 50 U / مل IL-2. </li><li style=";text-align:right;direction:rtl"> في يوم 7، كرر كما في الخطوة 4.6 ولكن استخدام IL-2 في اليوم من التحفيز، وليس IL-15. </li></ol> 5. ممثل النتائج ويصور A التخطيطي للبروتوكول GMP المتوافقة تصنيع وافقت عليها الهيئة في الشكل 1. تستغرق العملية حوالي 50 يوما. طرق نموذجية توليد خلايا محددة الفيروسات T توسيع القائمة من قبل الخلايا T الذاكرة، ولكن تفتقر إلى خلايا دم الحبل السري T فيروس ذوي الخبرة، وبالتالي نحن بحاجة الى رئيس الوزراء السابق فيفو T السذاجة الخلايا. للقيام بذلك، ونحن نستخدم الخلايا الجذعية وكذلك السيتوكينات IL-7، IL-12، وIL 15-،اللازمة لتوليد خصوصية الفيروسية. بعد 3 التحفيز، ينبغي أن يكون العائد على الخلايا 6 100x10. إذا الخلايا كافية ليست متاحة، يمكن إجراء التحفيز إضافية حتى العدد المرغوب فيه من CTLs متوفرة. ينبغي أن غالبية هذه الخلايا تكون CD3 + بمزيج من CD4 + + CD8 والخلايا التائية. ينبغي أن يكون هناك أقل من 15٪ خلايا NK (CD3-/CD56 +) وأقل من 1٪ CD19 + B الخلايا (الشكل 2). ينبغي أن تعترف CTL الموسع مستضد pp65 من CMV، وhexon بينتون من اتش، فضلا عن العديد من مولدات المضادات التي يتم التعبير EBV على EBV-LCL. عند اختباره في مقايسة ELISPOT، CTL ينبغي تفرز المزيد من الإنترفيرون؟ واستجابة لهذه المستضدات من المستضدات غير ذات صلة (الشكل 3). ينبغي أن CTL ليز أيضا الفيروسية الببتيد نابض هذه الأهداف التفجيرات PHA. في مقايسة 51 الإفراج الكروم، يجب CTL ليز LCL، CMVpp65-نابض واتش hexon و/أو أهداف نابض بينتون، نابض أهداف ولكن ليس مع الببتيدات غير ذي صلة (الشكل 4). <img src="/files/ftp_upload/3627/3627fig1.jpg" alt="الشكل 1."/> الشكل 1. الجيل متعددة للفيروسات محددة CTL من دم الحبل السري. التخطيطي تبين كامل عملية تصنيع CTL من دم الحبل السري. أولا يتم عزل الخلايا وحيدات النوى من دم الحبل السري الكسر 20٪ من وحدة دم الحبل السري السري مجزأة. باستثناء 5x10 6 خلايا التي يتم حفظها لتوليد LCL، ومطلي ثم جميع الخلايا الجذعية في وسائل الإعلام خلية لمدة 1-2 ساعات، وعند هذه النقطة ويتم حصاد الخلايا غير ملتصقة والمجمدة. ويتم تغذية DC DC ثم وسائل الإعلام التي تحتوي على IL-4 وGM-CSF. بعد 5 أيام من الثقافة، ونضجت وDC transduced مع ناقلات الغدانية تحتوي على مستضد CMV سائد مناعيا pp65. في البدء، يتم الجمع بين هذه DC مع الخلايا غير ملتصقة وكذلك السيتوكينات IL-7، IL-12،وIL-15. في التحفيز لاحقة، يتم استخدام ناقلات نفس الغدانية لتنبيغ EBV-LCL، والتي تستخدم الخلايا كما مستضد تقديم. IL-15 ويستخدم في التحفيز الثانية و2-IL بعد ذلك. <img src="/files/ftp_upload/3627/3627fig2.jpg" alt="الشكل 2."/> الشكل 2. النمط الظاهري الناتج من الخلايا T. أظهرت هو النسبة المئوية للخلايا الحية المدرجة في البوابة اللمفاويات. CTL + CD3 هي وCD4 + + CD8 أو CD3-/CD56- ولكن إلى حد كبير وCD19. <img src="/files/ftp_upload/3627/3627fig3.jpg" alt="الشكل 3."/> الشكل 3. وظائف T الخلية. تم اختبار T خصوصية الخلية عن طريق الإنترفيرون γELISPOT. ونابض خلايا T مع الببتيد متداخلة تغطي كامل البروتين من hexon، بينتون، pp65، ومستضد غير ذي صلة IE 1-. CTL وحده يشير إلى وسائل الإعلام وحدها. وLCL ذاتي المشع، وأضاف في 1x10 5 / أيضا. أظهرت هو المكان يعني تشكيل خلية جount الآبار ثلاث نسخ. <img src="/files/ftp_upload/3627/3627fig4.jpg" alt="الشكل 4."/> الشكل 4. النشاط لحل الخلايا من CTL. تم اختبار قدرة CTL مما أدى إلى التعبير عن أهداف ليز المستضدات الفيروسية في مقايسة الكروم 51. ونابض 51 الكروم، وصفت التفجيرات ذاتي PHA مع الببتيدات متداخلة تغطي كامل المستضد أو 51 الكروم، وصفت ذاتي cocultured LCL مع CTL. بعد 4 ساعات، وتحسب على الافراج غاما عداد غاما.

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Hematol. 91, 588 (2010).</li><li>Einsele, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+cytomegalovirus+(CMV)-specific+T+cells+for+the+treatment+of+CMV+infection+not+responding+to+antiviral+chemotherapy.">Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy.</a> Blood. 99, 3916 (2002).</li><li>Bao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+cytomegalovirus+pp65+and+IE-1+specific+cytotoxic+T+lymphocytes+for+cytomegalovirus-specific+immunotherapy+following+allogeneic+stem+cell+transplantation.">Expansion of cytomegalovirus pp65 and IE-1 specific cytotoxic T lymphocytes for cytomegalovirus-specific immunotherapy following allogeneic stem cell transplantation.</a> Biol. Blood Marrow Transplant. 14, 1156 (2008).</li><li>Shpall, E. J., Bollard, C. M., Brunstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cord+blood+transplant+therapies.">Novel cord blood transplant therapies.</a> Biol. Blood Marrow Transplant. 17, S39-S45 (2011).</li><li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+(G-Rex.">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex.</a> J. Immunother. 33, 305 (2010).</li></ol>

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<html> يمكن الاستراتيجيات الحالية التي تهدف إلى السيطرة على العدوى الفيروسية بعد CBT تكون فعالة، ولكن ترتبط مع أنها السميات كبيرة، غالية الثمن، ولا تمنح حماية ضد العدوى على المدى الطويل في وقت لاحق. في الواقع، قد يكون استخدام بعض الأدوية المضادة للفيروسات يحد من التوسع ف...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td></tr><tr><td> RPMI 1640 </td><td> إينفيتروجن </td><td> 21870-076 </td></tr><tr><td> DC وسائل الإعلام </td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA (متوسطة اضغط على) </td><td> ايرفين العلمية </td><td> 9195 </td></tr><tr><td> مصل الإنسان </td><td> الجوزاء بيو المنتجات </td><td> 100-110 </td></tr><tr><td> الغاز نفاذية Cultureware 18 </td><td> ويلسون، وولف </td><td> 80040S </td></tr><tr><td> IL-2 </td><td> تشيرون (TCH صيدلة) </td><td></td></tr><tr><td> IL-12 </td><td> NCI / CTEP </td><td></td></tr><tr><td> IL-15 </td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7 </td><td> R &amp; D </td><td> AFL207 </td></tr><tr><td> IL-1beta </td><td> R &amp; D </td><td> AFL201 </td></tr><tr><td> IL-6 </td><td> الخليةGenix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH الصيدلة </td><td></td></tr><tr><td> IL-4 </td><td> R &amp; D </td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-ألفا </td><td> R &amp; D </td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> BCM إنتاج النواقل CAGT مرفق </td><td></td></tr><tr><td> نقل البلازما مع مجموعة محول بالتركيبة الإناث </td><td> ميثاق الطبية </td><td> 89-550-66j </td></tr><tr><td> Lymphoprep </td><td> NYCOMED </td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

العدوى بالفيروس بعد زرع الخلايا الجذعية هي من بين الأسباب الأكثر شيوعا للوفاة، خاصة بعد دم الحبل السري (CB) زرع (CBT) حيث CB لا تحتوي على أرقام ملموس من الخلايا T فيروس ذوي الخبرة التي يمكن أن تحمي من الإصابة المتلقي 1 - 4 نحن وآخرون أظهرت أن الفيروس محددة CTL ولدت من المانحين مصليا وغرست إلى المستلم آمنة واقية. 5-8 ومع ذلك، وحتى وقت قريب، لم يكن الفيروس خلايا T محددة يتم إنشاء من دم الحبل السري، من المحتمل بسبب غياب خلايا محددة فيروس T الذاكرة. في محاولة لتقليد بشكل أفضل في ظروف فتيلة الجسم الحي من خلايا T السذاجة، أنشأنا الطريقة التي تستخدم الخلايا الجذعية المشتقة من CB (DC) transduced مع ناقلات الغدانية (Ad5f35pp65) التي تحتوي على مستضد CMV سائد مناعيا pp65، والقيادة وبالتالي خصوصية T الخلية نحو CMV واتش. [9] في البدء، ونحن نستخدم هذه أماهtured البلدان النامية وكذلك CB-T في الخلايا المشتقة وجود السيتوكينات IL-7، IL-12، وIL 15-10 الساعة تحفيز الثانية التي تستخدم EBV-تحولت الخلايا B، أو EBV LCL، التي تعبر عن كل من كامنة والتحللي مستضدات EBV. وتستخدم Ad5f35pp65-transduced EBV-LCL لتحفيز خلايا T في حضور IL-15 في تحفيز الثانية. التحفيز لاحقة استخدام Ad5f35pp65-transduced EBV-LCL وIL-2. من الخلايا وحيدات النوى 50x10 6 CB نحن قادرون على توليد ما يزيد عن 150 × 10 خلايا الفيروسات محددة 6 T ليز أن مستضد نابض الأهداف والسيتوكينات الإفراج ردا على التحفيز المستضدية 11 هذه الخلايا تم تصنيعها بطريقة GMP المتوافقة فقط باستخدام وقد ترجمت الكسر 20٪ من وحدة دم الحبل السري ومجزأة للاستخدام السريري.

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

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

نحن هنا وصف أول ممارسة التصنيع الجيد (GMP) المتوافقة مع طريقة لانتاج الفيروسات محددة الخلايا الليمفاوية T السامة للخلايا (CTL) من دم الحبل السري، ومصدرا للخلايا T السذاجة في الغالب.

توسيع الخلايا اللمفاوية T السامة للخلايا من دم الحبل السري أن الفيروس المضخم للخلايا الهدف، فيروس ابشتاين بار، واتش

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

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Research

JoVE Journal

Immunology and Infection

12.7K Views.  Baylor College of Medicine. نحن هنا وصف أول ممارسة التصنيع الجيد (GMP) المتوافقة مع طريقة لانتاج الفيروسات محددة الخلايا الليمفاوية T السامة للخلايا (CTL) من دم الحبل السري، ومصدرا للخلايا T السذاجة في الغالب.

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

إعداد واستخدام HIV - 1 CD4 + T الابتدائية المصابة خلايا والخلايا المستهدفة في الخلايا القاتلة الطبيعية فحوصات السامة للخلايا

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

طرق لتقييم موت الخلايا بيتا وساطة من قبل الخلايا اللمفاوية التائية السامة

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

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

إدخال إجهاد القص في دراسة الالتصاق الجرثومي

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

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

إنشاء فيروس ابشتاين بار النمو وتحولت خطوط الخليوي Lymphoblastoid

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

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.

السريرية تطبيق<em&gt; الجمال النائم</em&gt; ومستضد تقديم الخلايا الاصطناعية لتعديل وراثيا خلايا T من دم الحبل السري والمحيطية

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 ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

عزل مجموعات فرعية B-الخلية السلائف من دم الحبل السري

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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.

العزلة والتمايز TH17 من السذاجة CD4 T الخلايا اللمفاوية

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 ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

وضع النطاقي الحماقي الفيروسات محددة بوساطة خلية الحصانة ELISPOT الفحص لتقييم الإنترفيرون γ-عقب الحبل السري زرع الدم

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

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.

توليد خلايا T الإنسان مستضد خيفي محددة من الدم المحيطي

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

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.

شعاعي التنقل والسامة للخلايا وظيفة من فيروسات الرجعية تكرار ناقل Transduced، غير ملتصقة Alloresponsive T اللمفاويات

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