وينقسم البروتوكول لتوليد خلايا B التي تنشط من CD40 PBMC إلى جزأين : الجزء A يدل على إعداد CD40 يجند NIH/3T3 معربا عن الخلايا ، والتي سوف تستخدم في الخلايا المغذية لوحة محدد. الجزء باء يصف الفعلية CD40 - B الثقافة. A. إعداد الخلايا المغذية (tCD40L NIH/3T3) وNIH/3T3 tCD40L هو ملتصق الخلية الليفية خط الفئران ، والتي لا ينبغي أبدا أن تصبح متكدسة تماما. وبالتالي فإن الخلايا splitted مرتين في الأسبوع. لا ينصح زراعة على مدى أكثر من 6 أسابيع. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة المتوسطة القديمة من ثقافة الأولية مع ماصة معقمة وغسل الخلايا مع 10 مل من 1X PBS. نضح في برنامج تلفزيوني بعد الغسيل. </li><li style=";text-align:right;direction:rtl"> إضافة 4 مل التربسين / EDTA في قارورة 75 2 سم لمدة 5-10 دقائق عند 37 درجة مئوية. استخدام التنصت لطيف لفصل الخلايا. </li><li style=";text-align:right;direction:rtl"> إضافة 10 مل من النوع المتوسط ​​البرية ودوار بلطف. </li><li style=";text-align:right;direction:rtl"> نقل تعليق الخلية في أنبوب 50 مل مع ماصة معقمة وتدور في الخلايا أسفل نحو 225 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إزالة وطاف resuspend الكرية في 10 مل من النوع المتوسط ​​البرية. حساب عدد الخلايا من قسامة لتعليق الخلية وإعداد ثلاث أنابيب 50 مل مع عدد مناسب من الخلايا : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 1.5 × 10 6 خلايا لsubculturing </li><li style=";text-align:right;direction:rtl"> 0.2 × 10 6 خلية / جيد لتشعيع المستخدمة لزراعة الخلايا CD40 - B </li><li style=";text-align:right;direction:rtl"> تبقى لتجميد (عند الحاجة). </li></ol></li><li style=";text-align:right;direction:rtl"> تدور في الخلايا أسفل نحو 225 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إزالة طاف. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لsubculturing : resuspend 1.5 × 10 6 خلايا في المتوسط ​​10 مل النوع البري في 75 سم 2 خلية قارورة الثقافة (1.5 × كثافة الخلية 10 5 خلية / مل) ، إضافة G - 418 [0.7 ملغ / مل] ، واحتضان الخلايا في 37 درجة مئوية مع نسبة 5 ٪ CO 2. انقسام الخلايا مرتين في الأسبوع. </li><li style=";text-align:right;direction:rtl"> لزراعة الخلايا CD40 - B : تحتاج 1.2 × 10 6 خلايا لوحة 6 - جيدا واحدة. Resuspend الخلايا في المتوسط ​​النوع البري في مناطق ذات كثافة تبلغ 0.1 X 6 10 الخلايا / مل ، وأشرق لهم في 78 غراي. لوحة 2 مل من تعليق خلية في كل بئر واحتضان لهم عند 37 درجة مئوية مع نسبة 5 ٪ CO 2. استخدام هذه اللوحات المعدة لتنشيط خلايا B عندما tCD40L NIH/3T3 الخلايا هي ملتصقة (على الأقل 4 ساعات : التحقق من الالتزام مع المجهر ، لا تنتظر أكثر من 24 ساعة لبدء الخلية B - التحفيز). (متابعة باء) </li></ol></li></ol> B. B - 40 CD خلية ثقافة أولا إعداد PBMCs لتحفيز - CD40 (يوم 0) : يرجى ملاحظة : قبل متابعة التأكد من أن الخلايا المغذية هي ملتصقة. إضافة جديدة من الحلول دائما على الفور الى وسط النمو انترلوكين 4 و السيكلوسبورين قبل الاستخدام. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأخذ PBMCs ، سواء الطازجة أو إذابة مناسب. Resuspend PBMCs مرتين في برنامج تلفزيوني من 50mL 1X لغسلها وتدور باستمرار أول مرة في 265 x ج لمدة 7 دقائق والمرة الثانية في XG 190 لمدة 7 دقائق لإزالة الخلايا الأخرى. تجاهل وطاف resuspend الخلايا في 20 مل من برنامج تلفزيوني. تحديد عدد الخلايا في قسامة لتعليق الخلية. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار المبلغ المطلوب من الخلايا نحو 225 x ج لمدة 5 دقائق. لوحة 6 - جيدا 4 × 10 6 خلية / كذلك هناك حاجة ، وبالتالي 24 × 10 6 خلية لكل لوحة. </li><li style=";text-align:right;direction:rtl"> إزالة وطاف في resuspend PBMC في الخلايا 1 × 6 10 / مل في CD40 - B مستنبت استكملت حديثا مع 50 يو / مل من انترلوكين 4 كعامل نمو والسيكلوسبورين 0.63 ميكروغرام / مل وثمرة لمنع الخلايا التائية (تركيزات نظرا تشير إلى واحد مل مستنبت!). </li><li style=";text-align:right;direction:rtl"> إزالة طاف من 6 جيدا قبل لوحة المحتضنة مع الخلايا NIH/3T3 tCD40L. </li><li style=";text-align:right;direction:rtl"> غسل ملتصقة tCD40L NIH/3T3 الخلايا مع PBS مل 2 في البئر الأولى والخطوة الثانية مع 2 مل من CD40 - B المتوسطة الغسيل. </li><li style=";text-align:right;direction:rtl"> إضافة بلطف 4 مل من تعليق PBMC (1 × 10 6 خلية / مل) إلى كل جانب من لوحة 6 - جيدا. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا عند 37 درجة مئوية مع نسبة 5 ٪ CO 2. </li><li style=";text-align:right;direction:rtl"> في يوم 7 ، reculture الخلايا (3.2 متابعة). </li></ol> II. إعادة الزراعة من CD40 الخلايا البائية (يوم 7 أيام ومن ثم كل 3-4) : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مجموعات حصاد CD40 - B - 6 خلايا من جانب لوحة عن طريق إعادة التعليق مع ماصة 10 مل وتجمع لهم في أنبوب 50 مل. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار نحو 225 x ج لمدة 7 دقائق واستبدالها تماما طاف مع CD40 - B المتوسطة الغسيل. في حين عد كمية من الخلايا قسامة ، وتدور في الخلايا أسفل نحو 225 x ج لمدة 5 دقائق. Resuspend خلايا CD40 CD40 - B - B في مستنبت بتركيز 1 × 10 6 خلية / مل. </li><li style=";text-align:right;direction:rtl"> إضافة حلول جديدة من 4 انترلوكين في تركيز 50 يو / مل و 0.63 ميكروغرام / مل السيكلوسبورين ألف للمتوسط. </li><li style=";text-align:right;direction:rtl"> إزالة طاف من لوحة 6 - جيدا قبل المحتضنة مع الخلايا NIH/3T3 tCD40L. </li><li style=";text-align:right;direction:rtl"> غسل خلايا ملتصقة مع PBS مل 2 في البئر الأولى والخطوة الثانية مع 2 مل من CD40 - B المتوسطة الغسيل. </li><lط&gt; برفق إضافة 4 مل (1 × 10 6 خلية / مليلتر) من تعليق CD40 - B على كل جانب من لوحة 6 - جيدا. </li><li style=";text-align:right;direction:rtl"> احتضان لوحات عند 37 درجة مئوية مع نسبة 5 ٪ CO 2. </li><li style=";text-align:right;direction:rtl"> خلايا فرعية في كل 3-4 أيام مرة أخرى في نهاية المطاف مع نقية للغاية خلايا B - CD40 تفعيلها بعد ما مجموعه 14 يوما. </li></ol> جيم حل المشاكل -- ماذا لو CD40 - B الخلايا لا تنمو؟ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> هل راجعت للتعبير يجند CD40 الخلايا المغذية؟ </li><li style=";text-align:right;direction:rtl"> وينبغي لوحة متجهة الخلايا المغذية المستخدمة في التحفيز لا يكون أقدم من 24H؟ </li><li style=";text-align:right;direction:rtl"> هو تلوث محتمل مع الميكوبلازما؟ </li><li style=";text-align:right;direction:rtl"> كان الحل انترلوكين - 4 المستخدمة لإذابة مكملات طازجة وكان النشاط المناسب البيولوجي؟ </li><li style=";text-align:right;direction:rtl"> وأضاف السيكلوسبورين A في التركيز الصحيح؟ </li></ol><img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="الشكل 1" /> الشكل 1. الرجاء <a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">النقر هنا</a> لنسخة أكبر من الرقم 1. <img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="الشكل 2" /> الشكل 2. الرجاء <a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">النقر هنا</a> لنسخة أكبر من الشكل 2.

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25, 659-664 (2004).</li><li>Bergwelt-Baildon, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107, 2786-2789 (2006).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> Blood. 85, 1903-1912 (1995).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63, 2836-2843 (2003).</li><li>von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99, 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155, 249-256 (2009).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15, 955-965 (2008).</li></ol>

A. إعداد وسائل الإعلام : <table border="1"cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="2"> التغذية المتوسطة الخلية نوع البرية </td><td colspan="2"> اختيار الخلية المتوسطة المغذية </td></tr><tr><td> الكاشف </td><td> تركيز </td><td> الكاشف </td><td> تركيز </td></tr><tr><td> DMEM - هام ل/ F12 </td><td></td><td> DMEM - هام ل/ F12 </td><td></td></tr><tr><td> L - الجلوتامين </td><td> 365 ميكروغرام / مل </td><td> L - الجلوتامين </td><td> 365 ميكروغرام / مل </td></tr><tr><td> FBS </td><td> 10 ٪ </td><td> FBS </td><td> 10 ٪ </td></tr><tr><td> HEPES </td><td> 10 ملي </td><td> HEPES </td><td> 10 ملي </td></tr><tr><td> الجنتاميسين </td><td> 15 ميكروغرام / مل </td><td> الجنتاميسين </td><td> 15 ميكروغرام / مل </td></tr><tr><td></td><td></td><td> G - 418 </td><td> 0.7 ملغ / مل </td></tr></table><table cellspacing="0" border="1" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="2"> CD40 باء غسل المتوسطة </td><td colspan="2"> B - CD40 مستنبت </td></tr><tr><td> الكاشف </td><td> تركيز </td><td> الكاشف </td><td> تركيز </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td> </td><td> IMDM </td><td> </td></tr></tbody><tbody><tr><td> L - الجلوتامين </td><td> 584 ميكروغرام / مل </td><td> L - الجلوتامين </td><td> 584 ميكروغرام / مل </td></tr></tbody><tbody><tr><td> HEPES </td><td> 25 ملم </td><td> HEPES </td><td> 25 ملم </td></tr></tbody><tbody><tr><td> الجنتاميسين </td><td> 15 ميكروغرام / مل </td><td> الجنتاميسين </td><td> 15 ميكروغرام / مل </td></tr></tbody><tbody><tr><td> </td><td> </td><td> RH ترانسفيرين </td><td> 50 ميكروغرام / مل </td></tr></tbody><tbody><tr><td> </td><td> </td><td> RH الأنسولين </td><td> 5 ميكروغرام / مل </td></tr></tbody><tbody><tr><td> </td><td> </td><td> AB - Humanserum </td><td> 10 ٪ </td></tr></tbody></table> B. متنوعة الكواشف : <table cellspacing="0" border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> الكاشف </td><td> شركة </td><td> لكي لا </td></tr><tr></tr><tr><td> DMEM / هام لF12 </td><td> PAA </td><td> لا القط : E15 - 813 </td></tr><tr><td> IMDM </td><td> Invitrogen Gibco ® </td><td> REF 21980-032 </td></tr><tr><td> Dulbecco في برنامج تلفزيوني (10X) </td><td> PAA </td><td> القط لا H15 - 011 </td></tr><tr><td> AB - مصل الإنسان </td><td> Invitrogen </td><td> القط لا 34005100 </td></tr><tr><td> هولو ، ترانسفيرين الإنسان </td><td> سيغما </td><td> القط لا T0665 </td></tr><tr><td> الأنسولين البشري </td><td> سيغما </td><td> القط لا I2643 </td></tr><tr><td> Gencin ® </td><td> حدد الدلتا </td><td> الفن لا يوجد 7395800 </td></tr><tr><td> الإنسان إينترليوكين - 4 المؤتلف </td><td> Immunotools </td><td> القط لا 11130045 </td></tr><tr><td> السيكلوسبورين A </td><td> شركة نوفارتيس </td><td> القط لا 0078-0109-01 NDC </td></tr><tr><td> FBS </td><td> LONZA </td><td> القط لا DE14 - 802C </td></tr><tr><td> HEPES العازلة </td><td> PAA </td><td> القط لا S11 - 001 </td></tr><tr><td> G - 418 كبريتات </td><td> PAA </td><td> القط لا P02 - 012 </td></tr><tr><td> التربسين / EDTA (10X) </td><td> Invitrogen Gibco ® </td><td> REF 15400-054 </td></tr></tbody></table> جيم لوازم متنوعة : <table cellspacing="0" border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> الكاشف </td><td> شركة </td><td> لكي لا </td></tr><tr></tr><tr><td> ماصة معقمة نصائح </td><td> Sarstedt </td><td></td></tr><tr><td> 6 كذلك لوحة </td><td> نونك </td><td> القط لا 140675 </td></tr><tr><td> 50mL أنبوب مخروطي </td><td> دينار بحريني فالكون ™ </td><td> القط لا 352070 </td></tr><tr><td> نسيج الثقافة القارورة </td><td> SARSTEDT </td><td> القط لا 83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

وقد تم تحديد CD40 تنشيط خلايا B (CD40 - B الخلايا) كمصدر بديل للالمناعية تنشيطية مستضد تقديم الخلايا ، 1-3 (APC) لعلاج مناعي السرطان. بالمقارنة مع الخلايا الجذعية (DCS) ، وأفضل APC تتسم ، CD40 - B وخلايا عدة متميزة الخصائص البيولوجية والتقنية. مماثلة للبلدان النامية ، تظهر الخلايا B تعبير زيادة MHC وشارك في تنشيطية جزيئات (Fig.1b) ، المعرض قدرة قوية المهاجرة وتقديم العرض مستضد بكفاءة إلى خلايا T ، بعد التحفيز مع يجند انترلوكين 4 و CD40 (CD40L). لكن ، على النقيض من البلدان النامية غير ناضجة أو ناضجة ، CD40 - B الخلايا التعبير عن العقدة الليمفاوية ثالوث صاروخ موجه كامل يتكون من CD62L ، CCR7/CXCR4 وظيفة الكريات البيض مستضد - 1 (LFA1 ، CD11a/CD18) ، اللازمة لصاروخ موجه إلى الأجهزة اللمفاوية الثانوية (Fig.1a) 3. يمكن أن تتولد CD40 - B الخلايا دون صعوبات من كميات صغيرة جدا من الدم المحيطي التي يمكن مزيد من التوسع في المختبر لكميات كبيرة جدا من درجة عالية من محض CD40 - B الخلايا السرطانية (&gt; 10 9 خلايا لكل مريض) من المتبرعين الأصحاء وكذلك مرضى (Fig.1c ، د) 1،4. في هذا البروتوكول ونحن لشرح كيفية الحصول على تفعيلها بشكل كامل CD40 - B الخلايا من PBMC الإنسان. الجزيئات الأساسية للثقافة الخلية هي CD40 يجند ، انترلوكين 4 (IL - 4) ، والسيكلوسبورين A (CSA) ، والتي تتجدد في حلقة اليوم ثقافة 3-4 لأغراض المختبر قدمت CD40 - تحفيز الخلايا التي NIH/3T3 معربا عن المؤتلف الإنسان CD40 يجند (tCD40L NIH/3T3) 5. لتجنب التلوث مع المنظمات غير transfected الخلايا ، والتعبير ليجند CD40 الإنسان على transfectants يجب أن يكون محددا بشكل منتظم (Fig.2). بعد 14 يوما CD40 B - الثقافات الخلية تتكون من خلايا أكثر من 95 ٪ ب نقية وتوسيع CD40 - B الخلايا أكثر من 65 يوما هو ممكن في كثير من الأحيان من دون أي خسارة وظيفة 1 و 4. CD40 - B الخلايا تأخذ بكفاءة أعلى ، و 6 مولدات المضادات العملية الحالية لخلايا تي. انهم لا naϊve فقط الوزراء ، وإنما أيضا توسيع الخلايا التائية الذاكرة 7،8. ويمكن استخدام CD40 تنشيط خلايا B لدراسة الخلية B - التنشيط ، والتمايز وظيفة. وعلاوة على ذلك ، لأنها تمثل أداة واعدة للقاح علاجي أو وقائي ضد الأورام 9.

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells at JoVE.com

Generation of Human CD40-activated B cells

في هذا الفيديو نقدم<em&gt; خارج الحي</em&gt; توليد والتوسع في خلايا B - CD40 تنشيط (CD40 - B) من خلايا الدم المحيطية وحيدات النوى (PBMC) عن طريق التحفيز مع CD40 يجند وانترلوكين 4.

جيل من الخلايا البشرية باء تنشيط CD40

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

generation-of-human-cd40-activated-b-cells

Research

JoVE Journal

Biology

15.2K Views.  University Hospital of Cologne, Department I of Internal Medicine. في هذا الفيديو نقدم خارج الحي توليد والتوسع في خلايا B - CD40 تنشيط (CD40 - B) من خلايا الدم المحيطية وحيدات النوى (PBMC) عن طريق التحفيز مع CD40 يجند وانترلوكين 4.

Video: Generation of Human CD40-activated B cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

الفئران نموذج تفعيل CD40 - B من الخلايا

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Immunology and Infection

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

المؤتلف إنتاج فيروسات الرجعية والعدوى من خلايا B

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

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 ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

الجيل المؤتلف الإنسان مفتش حيدة النسيلة من مخلد خلايا التصنيف B

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

العزلة وتنشيط الخلايا الليمفاوية الفئران

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

توليد<em&gt; دي نوفو</em&gt; محددة مستضد تي الإنسان مستقبلات الخلية من قبل فيروسات تنبيغ من مركزية Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

جيل من الوحيدات المستمدة الخلايا الجذعية البشرية من الدم الكامل

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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

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

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

جيل من التمييزية مونوكلونل البشرية من خلايا نادرة محددة مستضد ب المتداولة في الدم

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

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

في المختبر التمايز نموذج الذاكرة البشرية العادية ب الخلايا إلى خلايا البلازما المعمرة

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...