وأيد هذا العمل من قبل المعاهد الوطنية للصحة CA104757 المنحة R01 (MH Manjili). نحن نعترف بامتنان دعم مركز سرطان سي يو ماسي ومؤسسة الكومنولث للبحوث السرطان.

1. عزل الخلايا اللمفاوية 5 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ورم الغدد الليمفاوية عزل تجفيف أو الطحال من الفئران الحاملة للورم FVBN202 المعدلة وراثيا وإعداد وحيدة الخلية في تعليق الجليد الباردة RPMI1640 تستكمل مع FBS 10 ٪. B / I التنشيط في 50 مل من أنابيب البولي بروبلين النتائج المخروطية في زيادة الغلة الخلايا التائية مقارنة أنابيب البوليسترين. ويتم حقن كيتامين زيلازين الملكية الفكرية للتخدير. يستخدم خلع عنق الرحم كوسيلة من وسائل القتل الرحيم. </li><li style=";text-align:right;direction:rtl"> ثقافة الخلايا (10 6 خلية / مل) في المتوسط ​​كاملة تحتوي على 15 ٪ FBS مع bryostatin 1 - 5 (نانومتر) وionomycin (1 ميكرومتر) جنبا إلى جنب مع 80 يو / مل من IL - 2 (Peprotech) لمدة 16 ساعة. </li><li style=";text-align:right;direction:rtl"> غسل خلايا ثلاث مرات مع المتوسط ​​الدافئة (37 درجة مئوية) والثقافة في الخلايا 6 10 / مل في المتوسط ​​الكامل مع IL - 7 (10 نانوغرام / مل) و IL - 15 (10 نانوغرام / مل) (Peprotech) لمدة 24 ساعة . </li><li style=";text-align:right;direction:rtl"> نبض الخلايا مع IL - 2 (40 يو / مل) لمدة 24 ساعة. </li><li style=";text-align:right;direction:rtl"> انقسام الخلايا والثقافة لهم IL - 7 و IL - 15 (10 نانوغرام / مل) لمدة 4 ايام. تغيير متوسطة وانقسام الخلايا إذا لزم الأمر كل أيام 2. </li></ol> 2. توسيع تحديد طية من الخلايا T التي كتبها عدد الخلايا وتحليل التدفق الخلوي 5 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عدد الخلايا التي المجهر الضوئي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعد مناسبة التخفيف الخلية (1:100) في الزرقاء التريبان وإضافة ميكرولتر قليلة على عدادة الكريات </li><li style=";text-align:right;direction:rtl"> 9 مربعات العد وتحديد مجموع عدد الخلايا بقسمة عدد الخلايا إلى عدد من الغرف مضروبا في عامل التخفيف. إلا أن النتائج الحالية رقم 4 × 10 خلية / مل. </li></ol></li><li style=";text-align:right;direction:rtl"> تحديد نسبة CD8 CD4 + + وخلايا تي في الخلايا عن طريق توسيع التدفق الخلوي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استكملت كتلة غير محددة وملزمة من الأجسام المضادة لمستقبلات القطعة Fc بواسطة زراعة الخلايا مع anti-CD16/CD32 الأضداد (Biolegend) لمدة 20 دقيقة على الجليد وغسل الخلايا ثم مرتين مع 2 مل من الجليد الباردة مع PBS أزيد الصوديوم 1 ٪ . </li><li style=";text-align:right;direction:rtl"> وصمة عار على زراعة الخلايا مع FITC - CD4 والأضداد PE - CD8 لمدة 20 دقيقة على الجليد وغسل الخلايا ثم مرتين مع 2 مل من الجليد الباردة PBS تستكمل مع FBS 1 ٪ و 0.1 ٪ أزيد الصوديوم. </li><li style=";text-align:right;direction:rtl"> تحديد الخلايا مع بارافورمالدهيد 1 ٪ وتشغيل عينات على بيكمان كولتر FC 500 و تحليل استخدام الإصدار 4.3 برنامج القمة. </li></ol></li></ol> 3. الموسع تحديد الورم خصوصية فيفو السابقين الخلايا T <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ثقافة توسيع فيفو السابقين الخلايا الليمفاوية في المتوسط ​​الكامل في نسبة 10:01 العصبية الإيجابية مع الخلايا السرطانية المشع MMC (15000 RAD) لمدة 24 ساعة 5 </li><li style=";text-align:right;direction:rtl"> supernatants الحصاد وتخزينها في -80 درجة مئوية حتى المستخدمة. 5،6 </li><li style=";text-align:right;direction:rtl"> كشف الإنترفيرون γ باستخدام الماوس الإنترفيرون γ ELISA تعيين دينار بحريني (Pharmingen) وفقا لبروتوكول الشركة المصنعة. 5،6 </li></ol> 4. تحديد المضادة للورم وظيفة خلايا تي فيفو السابقين الموسع 5،6 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان خلايا T مع الخلايا السرطانية في 10:01 المستجيب : النسب المستهدفة لمدة 48 ساعة في المتوسط ​​الكامل في المتوسط ​​3 مل كاملة (RPMI - 1640 تستكمل مع 100U / مل من البنسلين ، 100μg / الستربتوميسين مل ، 10 ٪ FBS ، والجلوتامين β -- المركابتويثانول) و20U / مل من IL - 2 (Peprotech) في 6 اطباق الثقافة جيدا 37 ° C CO / 5 ٪ 2. </li><li style=";text-align:right;direction:rtl"> أداء ثلاث الضد اللون لتلوين العصبونات (anti-c-Erb2/c-neu ، استنساخ - 4 ، Calbiochem) ، يليه مفتش الماوس PE المضادة ، Annexin V - FITC والتأيد Propidium (PI) وفقا لبروتوكول الشركة المصنعة دينار بحريني (Pharmingen) </li><li style=";text-align:right;direction:rtl"> على الخلايا العصبية بوابة ورم إيجابي وتحليل جدوى (Annexin V-/PI-) من الخلايا السرطانية </li></ol> 5. الفأر نموذج من سرطان الثدي ويمكن استخدام إناث الفئران المعدلة وراثيا FVBN202 (مختبرات نهر تشارلز) عن مصدر الخلايا التائية الورم التفاعل. هذه الفئران overexpress an unactivated الفئران العصبية التحوير في إطار تنظيم MMTV المروج ، ونتيجة لسرطان الثدي تطوير عفوية بين 4-10 شهرا من العمر 7. تطوير هذه الفئران قبل تحولها الى تضخم الثدي مماثلة لسرطان الأقنية في الموقع (DCIS) قبل وضع carcinoma8 عفوية. وتستخدم عفوية الفئران الحاملة للورم والجهات المانحة من خلايا تي. 6. ممثل النتائج : تنشيط خلايا T مع B / I عن النتائج 16 ساعة في قتل الخلايا التائية السذاجة التي لا يتم توعيتهم مع الورم في الجسم الحي. بعد الانتقائية B / الأول من الخلايا التائية الورم التفاعل أنهم توسيع تصل إلى 2.8 أضعاف في غضون 6 ايام الثقافة مع السيتوكينات سلسلة غاما (الشكل 1). كلا CD8 + و + CD4 يتم توسيع الخلايا التائية بالتساوي مع السيتوكينات سلسلة غاما (الشكل 2). فيفو السابقين الخلايا T - الموسعة تظهر استجابة عالية ضد الأورام التي كانت الفئران المانحة توعيتهم ، وتقييمها من قبل لإنتاج الإنترفيرون γ في وجود سرطان الثدي إيجابي العصبية الماوس (MMC) خلايا الورم (الشكل 3). يمكن توسيع فيفو السابقين الخلايا التائية تحفز الخلايا العصبية الإيجابية في سل الورم MMCليرة سورية بحيث بقاء الخلايا السرطانية تنخفض من 92 ٪ إلى 61 ٪ في غضون 48 ساعة (الشكل 4). <img src="/files/ftp_upload/2381/2381fig1.jpg" alt="الشكل مفقود" /> الشكل 1. توسيع الغطاء من الخلايا الليمفاوية في نقاط زمنية مختلفة التالية B / I التنشيط (يوم 1) ، وبحكم التوسع المجراة مع السيتوكينات سلسلة غاما (أيام 3 و 5 و 7) <img src="/files/ftp_upload/2381/2381fig2.jpg" alt="الشكل مفقود" /> الشكل 2. نسبة مجموع CD4 + و CD8 + الخلايا التائية قبل وبعد التوسع لمدة 7 أيام مع السيتوكينات سلسلة غاما. <img src="/files/ftp_upload/2381/2381fig3.jpg" alt="الشكل مفقود" /> الشكل 3. الورم تحفيز الإنترفيرون γ إنتاج خلايا تي المعزولة من الفئران الحاملة للورم قبل وبعد التوسع لمدة 7 أيام مع السيتوكينات سلسلة غاما ، وذلك باستخدام الإنترفيرون γ ELISA <img src="/files/ftp_upload/2381/2381fig4.jpg" alt="الشكل مفقود" /> الشكل 4. ظيفة السامة للخلايا في الجسم الحي السابقين توسيع الخلايا التائية مع السيتوكينات سلسلة غاما ضد سرطان الثدي إيجابي العصبية الماوس (MMC) الخلايا السرطانية

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	<li>Goodell, V., Waisman, J., Salazar, L. G., de la Rosa, C., Link, J., Coveler, A. L., Childs, J. S., Fintak, P. A., Higgins, D. M., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Level+of+HER-2/neu+protein+expression+in+breast+cancer+may+affect+the+development+of+endogenous+HER-2/neu-specific+immunity.">Level of HER-2/neu protein expression in breast cancer may affect the development of endogenous HER-2/neu-specific immunity.</a> Mol Cancer Ther. 7, 449-454 (2008).</li><li>Disis, M. L., Knutson, K. L., Schiffman, K., Rinn, K., McNeel, D. 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Immunother. 29, 284-293 (2006).</li><li>Bear, H. D., Roberts, J., Cornell, D., Tombes, M. B., Kyle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+of+cancer+with+pharmacologically+activated+lymph+node+lymphocytes:+a+pilot+clinical+trial.">Adoptive immunotherapy of cancer with pharmacologically activated lymph node lymphocytes: a pilot clinical trial.</a> Cancer Immunol Immunother. 5, 269-274 (2001).</li><li>Morales, J. K., Kmieciak, M., Graham, L., Feldmesser, M., Bear, H. D., Manjili, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+HER2/neu-specific+T+cells+expanded+with+alternating+gamma+chain+cytokines+mediate+tumor+regression+when+combined+with+the+depletion+of+myeloid-derived+suppressor+cells.">Adoptive transfer of HER2/neu-specific T cells expanded with alternating gamma chain cytokines mediate tumor regression when combined with the depletion of myeloid-derived suppressor cells.</a> Cancer Immunol Immunother. 58, 941-953 (2009).</li><li>Cha, E., Graham, L., Manjili, M. H., Bear, H. D., Guy, C. T., Webster, M. A., Schaller, M., Parsons, T. J., Cardiff, R. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Danger+signals+and+nonself+entity+of+tumor+antigen+are+both+required+for+eliciting+effective+immune+responses+against+HER-2/neu+positive+mammary+carcinoma:+implications+for+vaccine+design.">Danger signals and nonself entity of tumor antigen are both required for eliciting effective immune responses against HER-2/neu positive mammary carcinoma: implications for vaccine design.</a> Cancer Immunol Immunother. 57, 1391-1398 (2008).</li><li>Stern, J. B., Smith, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-2+induction+of+T-cell+G1+progression+and+c-myb+expression.">Interleukin-2 induction of T-cell G1 progression and c-myb expression.</a> Science. 233, 203-206 (1986).</li><li>Kittipatarin, C., Khaled, A. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubation+of+antigen-sensitized+T+lymphocytes+activated+with+bryostatin+1+++ionomycin+in+IL-7+++IL-15+increases+yield+of+cells+capable+of+inducing+regression+of+melanoma+metastases+compared+to+culture+in+IL-2.">Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2.</a> Cancer Immunol Immunother. 58, 1565-1576 (2009).</li></ol>

الإعلان عن أي تضارب في المصالح.

ويمكن تحقيق التوسع الانتقائي للورم خلايا تي التفاعل مع وظيفة مضادة للورم المستجيب من البروتوكول المقترح باستخدام B / I التنشيط وبحكم التوسع المجراة مع السيتوكينات سلسلة غاما IL - 2 ، IL - 7 و IL - 15. بينما IL - 2 هو عامل نمو الخلايا التائية التي يمكن أن تدعم التمايز وا?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bryostatin 1</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B7431-10ug</td>
<td>&nbsp;</td>
<tr>
<td>Ionomycin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>407950</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-7</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>217-17</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-15</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>210-15</td>
<td>&nbsp;</td>
<tr>
<td>Human IL-2</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>200-02</td>
<td>&nbsp;</td>
<tr>
<td>RPMI1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Gemini</td>
<td>100-106</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
<tr>
<td>L- glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<td>&beta;- mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>anti-CD16/32 antibody</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>101302</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-FITC Apoptosis Detection Kit</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>556547</td>
<td>&nbsp;</td>
<tr>
<td>FITC-CD4</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100406</td>
<td>&nbsp;</td>
<tr>
<td>PE-CD8</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100708</td>
<td>&nbsp;</td>
<tr>
<td>anti-c-Erb2/c&#x2013;Neu</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>OP16</td>
<td>&nbsp;</td>
<tr>
<td>PE- anti mouse IgG</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>405307</td>
<td>&nbsp;</td>
<tr>
<td>formaldehyde</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>04018</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Hycor</td>
<td>87144</td>
<td>&nbsp;</td>
<tr>
<td>Light microscope</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>V200073</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IFN-&gamma; ELISA set</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>555138</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture flasks</td>
<td>&nbsp;</td>
<td>Greiner</td>
<td>658175</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

ex vivo expansion of tumor-reactive t cells by means of bryostatin 1/ionomycin and the common gamma chain cytokines formulation

أفادت الأنباء أن مرضى سرطان الثدي قد موجودة مسبقا الاستجابات المناعية ضد الأورام 1،2 بهم. ومع ذلك ، فشلت هذه الاستجابات المناعية لتوفير الحماية الكاملة ضد تطوير أو تكرار الاصابة بسرطان الثدي. للتغلب على هذه المشكلة عن طريق زيادة وتيرة ورم الخلايا التائية التفاعل ، واستخدمت العلاج المناعي بالتبني. وقد استخدمت مجموعة متنوعة من البروتوكولات لتوسيع خلايا الورم T - معينة. هذه البروتوكولات ، ومع ذلك ، وتقتصر على استخدام المضادات الورم فيفو السابقين لتنشيط خلايا T مستضد محدد. قريب جدا ، وقد استخدمت المشتركة السيتوكينات سلسلة غاما مثل IL - 21 IL - 2 ، IL - 7 ، IL - 15 ، وحدها أو في تركيبة من أجل تعزيز مكافحة الورم الاستجابات المناعية 3. ومع ذلك ، فإنه ليس واضحا ما سيعمل أفضل صياغة لتوسيع خلايا الورم T التفاعل. هنا نقدم بروتوكولا لتفعيل انتقائية وتوسيع التفاعل ورم الخلايا التائية من نموذج FVBN202 الماوس المعدلة وراثيا من سرطان الثدي HER-2/neu إيجابية للاستخدام في علاج الخلايا T بالتبني من سرطان الثدي. البروتوكول يتضمن تنشيط خلايا T مع bryostatin-1/ionomycin (B / I) و IL - 2 في غياب مستضدات الورم لمدة 16 ساعة. B / I التنشيط يقلد الاشارات بين الخلايا التي تؤدي إلى تنشيط الخلايا T عن طريق زيادة نشاط البروتين كيناز سي والكالسيوم داخل الخلايا ، على التوالي 4. هذا البروتوكول على وجه التحديد ينشط الخلايا T - ورم معين في حين قتل خلايا T لا صلة لها بالموضوع. يتم تربيتها في B / I - تنشيط خلايا T مع IL - 7 و IL - 15 لمدة 24 ساعة وبعد ذلك مع نابض IL - 2. بعد 24 ساعة ، ويتم غسل الخلايا التائية ، تقسيم ، والمثقف مع IL - 7 + IL - 15 ليوم 4 إضافية. يتم تحديد فعالية وخصوصية للورم المضادة للورم في خلايا الجسم الحي السابقين T الموسعة.

Watch this Scientific Journal Video about Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation at JoVE.com

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

بروتوكول للكفاءة<em&gt; خارج الحي</emيوصف&gt; التوسع في ورم الخلايا التائية التفاعل من ورم الغدد الليمفاوية تجفيف أو غيرها من الأنسجة اللمفاوية الثانوية الحاملة للورم المضيفين. يوسع هذا البروتوكول بشكل انتقائي خلايا T للورم محددة لاستخدامها في العلاج المناعي بالتبني من سرطان الثدي.

توسيع فيفو السابقين الخلايا T ورم التفاعل من قبل وسائل 1/Ionomycin Bryostatin وصياغة سلسلة غاما المشتركة السيتوكينات

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

ex-vivo-expansion-tumor-reactive-t-cells-means-bryostatin-1ionomycin

Research

JoVE Journal

Immunology and Infection

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

14.6K Views.  Virginia Commonwealth University- Massey Cancer Center. بروتوكول للكفاءة خارج الحي التوسع في ورم الخلايا التائية التفاعل من ورم الغدد الليمفاوية تجفيف أو غيرها من الأنسجة اللمفاوية الثانوية الحاملة للورم المضيفين. يوسع هذا البروتوكول بشكل انتقائي خلايا T للورم محددة لاستخدامها في العلاج المناعي بالتبن...

Video: Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA - الإيج بناء خلايا مولدة الاصطناعي تقديم الكفء لل خارج الحي توسيع CTL الإنسان

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

التصوير خارج الحي من خلايا تي في الفئران شرائح العقدة الليمفاوية مع Widefield ومجاهر متحد البؤر

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

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

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

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

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

التوسع في الدم المحيطي الإنسان خلايا T γδ باستخدام Zoledronate

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

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

مستضد تقديم الاصطناعي تنشيط الخلية بوساطة (AAPC) والتوسع في الخلايا التائية القاتلة الطبيعية

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

فصل<em&gt; المتصورة المنجلية</em&gt; في وقت متأخر مرحلة المصابين تبدي الكريات الحمر بوسائل المغناطيسي

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

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

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

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

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

تقييم الفطرية الاستشعار من HIV-1 CD4 المصابة<sup&gt; +</sup&gt; T الخلايا عن طريق الخلايا الجذعية بلازماوية الشكل باستخدام<em&gt; خارج الحي</em&gt; المشارك ثقافة النظام.

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

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

ل<em&gt; السابقين فيفو</em&gt; نموذج لهجوم T-خلية الموجه دبقية قليلة التغصن في الدماغ الحادة شرائح

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T خلايا التقاط البكتيريا التي كتبها Transinfection من الخلايا الجذعية

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

العزلة و<em&gt; السابقين فيفو</em&gt; ثقافة Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; خلايا T γδ، وهو Extrathymic αβT خلايا السلف

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

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

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

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