ويدعم هذا العمل في جزء من المعاهد الوطنية للصحة / NCATS جوائز العلوم السريرية وبالحركة لجامعة فلوريدا TL1 TR000066 وUL1 TR000064، ملحق التنوع من الأصل غرانت R01AI056152 من المعهد الوطني للصحة، منحة كاشف العلوم البيولوجية دينار بحريني، وجامعة من ولاية فلوريدا.

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لا الإفصاحات لتعلن.

هنا وصفناها بروتوكول لتحقيق التمايز في المختبر TH17. دراسة TH17 التمايز هو المهم، وتمايز الخلايا اللمفية تي في فرعية TH17 أمر بالغ الأهمية من أجل القضاء الفعال لمسببات الأمراض البشرية 13. على العكس، ارتبط إنتاج IL17 مع تطور المرض المناعة الذاتية 13. طريقة TH17 ا?...

CD4 + T الليمفاوية (خلايا تي) تلعب دورا حاسما في الدفاع المناعي بوساطة نظام ضد الكائنات الدقيقة المعدية. على العكس، كما يرتبط ارتباطا وثيقا مع خلايا T ظهور وتطور أمراض المناعة الذاتية مثل مرض السكري من النوع 1، الذئبة الحمامية، والتهاب المفاصل الروماتويدي. CD4 + الخلايا اللمفية تي تصبح تفعيلها من خلال مجموعة من مستقبلات الخلايا التائية (TCR) التفاعل مع مستضد وما شابه ذلك / مجمع التوافق النسيجي الرئيسي الثاني (MHCII) جزيئات، والتفاعلات مستقبلات CD28 مع B7.1/B7.2 بروابط 15. بالإضافة إلى توفير التحفيز TCR وCD28 شارك في التحفيز، والخلايا العارضة للمستضد أيضا توفير بيئة خلوى، والذي يحدد الدولة تمايز تي اللمفاويات، وبالتالي توجيه استجابة الخلايا اللمفاوية T لمستضد معين. التفاعلات الممرض متميزة / خلية مستضد تقديم خلق بيئات خلوى متميزة، والتي تحرف الخلايا اللمفية تي أسفل مسارات متميزة تركز على ايليmination من مسببات المرض الشروع. للأسف، T مسارات الخلايا اللمفاوية المستجيب، صممت في الأصل للقضاء على غزو مسببات الأمراض، ويمكن أن توجه ضد أنسجة خطأ الذاتي 15. وبالتالي، فهم أفضل من كل دولة التمايز متميزة CD4 + T الخلية فرعية أمر بالغ الأهمية لفهمنا لكيفية لتعديل التوازن بين القضاء على مسببات الأمراض والتسامح في تقرير المصير. بالإضافة إلى الاستجابة Th1، الاستجابة Th2، ومحرض T التنظيمية مسارات تمايز الخلايا، الخلايا الليمفاوية T السذاجة ويمكن أيضا أن تكون مدفوعة من قبل السيتوكينات أسفل مسار TH17. بينما TH1 الخلايا مسببات الأمراض بين الخلايا المقاتلة، وخلايا الاستجابة Th2 القضاء على مسببات الأمراض خارج الخلية، والخلايا التائية التنظيمية (Tregs) تقليل الاستجابات الالتهابية 1، 16؛ خلايا TH17 تلعب دورا هاما في القضاء على البكتيريا والفطريات خارج الخلية. يتم الرمز الخلايا TH17 عموما تعبير عن النسخ عامل النسب المحددة RORγT وإنتاج IL-17A، وهو ما يعزز تفعيل الضامة والعدلات 1، 7. قد تورطت في عدة خلايا TH17 اضطرابات المناعة الذاتية، ونماذج القوارض المرتبطة بها. على سبيل المثال، فقد ثبت أن IL-23 (الذي هو مطلوب للحفاظ على النمط الظاهري TH17)، ولكن، لم يكن IL-12 المذنب المركزي في التهاب الدماغ التجريبية المناعة الذاتية (EAE)، نموذج المرض القوارض لMS. وفيما بعد تبين أن التخفيضات في إنتاج IL-17 ترتبط بالوقاية EAE 2 و 6 و 17. علاوة على ذلك، وقد ارتبطت الخلايا TH17 الذين يعانون من اضطرابات المناعة الذاتية الأخرى بما في ذلك التهاب المفاصل والذئبة الحمامية الجهازية (SLE) 10، 16. IL-23 P19 ناقصة - / - عرضت الفئران لديها أعداد قليلة جدا من الخلايا TH17، ومقاومة للتطوير ليس فقط EAE، ولكن أيضا التهاب المفاصل الناجم عن الكولاجين، وهو نموذج لالتهاب المفاصل الروماتويدي 10، 18. بالإضافة إلى ذلك، الفئران التي عولجت مع تحييد الأجسام المضادة IL-17A الخلفإيه عثر على بداية التهاب المفاصل الناجم عن الكولاجين أيضا أن يكون القرار من تلف المفاصل 18. تجدر الإشارة إلى أن دور الخلايا TH17 في تطور أمراض المناعة الذاتية ويبقى أن توصف بأنها أظهرت الأبحاث الحديثة أيضا دورا وقائيا من الخلايا TH17 في النوع 1 من مرض السكري 9 و 11 و التهاب الأمعاء 14. تؤكد هذه الدراسات على أهمية TH17 التمايز في المناعة الذاتية. في المختبر TH17 التمايز هو وسيلة ضرورية في أبحاث الخلايا T لأن هناك اثنين على الأقل من الأسئلة المحيرة التي تتطلب مزيدا من التحقيق: 1) كيف بالضبط هل IL-6 تنظيم التوازن بين Treg وTH17 التمايز، و 2) ما هي الآليات الدقيقة وراء IL-17 التي يسببها الاضطرابات الالتهابية؟ لدينا وسيلة توظف خلايا CD4 + CD25-T من الطحال والغدد الليمفاوية من الفأرة C57BL / 6. من المهم أن نلاحظ أنه على الرغم من أنه من الممكن للحث على تمايز TH17 باستخدام النجاسةالسكان، والحصول على ما لا يقل عن 80٪ نقية CD4 + السكان الخلية CD25-T ينفي أي قلق من التلوث ويضمن نتائج TH17 التمايز أكثر نجاحا. من أجل تحقيق التمايز TH17 السليم، يتم تحضين الخلايا CD4 + CD25-T بحضور مكافحة CD3 ومكافحة CD28، والتي توفر إشارات التنشيط، 1 و 2 على التوالي، وIL-6، وTGF-β. على الرغم من أن تم الإبلاغ عن أن IL-23 وحده يمكن استخدامها لتحقيق TH17 التمايز، وقد تجلى ذلك في وقت لاحق أن IL-23 هو ضروري لاستقرار السكان الخلية TH17، ولكن IL-6 و TGF-β ضرورية لTH17 التمايز 3، 18، 19. وقد أظهرت الدراسات أن الفئران يتم التعبير عن مستقبلات IL-23 على خلايا CD4 + T فقط بعد أن تكون قد حفز مع IL-6 و TGF-β 13، 18. أيضا، سوف الخلايا TH17 تتطور بنجاح في حضور IL-23-منع الأجسام المضادة طالما IL-6 و TGF-β موجودة 18، 19. على هذا النحو، وهذا التمايز TH17 بروتوكول الأقليمايديس الظروف المناسبة للحث على TH17 التمايز بنجاح. تطوير فهم أفضل للآليات الأساسية TH17 التمايز وIL-17 إنتاج تقديم الفرصة لتطوير علاجات أفضل تهدف إلى اضطرابات المناعة الذاتية 13.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>Sterile Polyestrene Petri Dish</td>
 <td>Fisher Scientific</td>
 <td>0875713A</td>
 <td>60 mm x 15 mm</td>
 </tr>
 <tr>
 <td>autoMACS Running Buffer</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td></td>
 </tr>
 <tr>
 <td>Premium Microscope Slides, Frosted</td>
 <td>Fisher Scientific</td>
 <td>12-544-3</td>
 <td>3" x 1"1 mm</td>
 </tr>
 <tr>
 <td>5 ml 21G1 Latex Free Syringe and Needle</td>
 <td>BD Biosciences</td>
 <td>309632</td>
 <td></td>
 </tr>
 <tr>
 <td>Corning 15 ml Centrifuge Tubes</td>
 <td>Sigma-Aldrich</td>
 <td>CLS430791</td>
 <td></td>
 </tr>
 <tr>
 <td>Nylon 40 microns</td>
 <td>Miami Aquaculture</td>
 <td>Nylon 40/26</td>
 <td></td>
 </tr>
 <tr>
 <td>Microtest tissue culture plate 96 well U bottom </td>
 <td>BD Biosciences</td>
 <td>35-3077</td>
 <td></td>
 </tr>
 <tr>
 <td>Corning Costar 24 well cell culture plates</td>
 <td>Sigma-Aldrich</td>
 <td>CL3524</td>
 <td></td>
 </tr>
 <tr>
 <td>Eppendorf Tubes 1.5 ml</td>
 <td>Fisher Scientific</td>
 <td>05-408-129</td>
 <td></td>
 </tr>
 <tr>
 <td>Purified NA/LE Hamster Anti-Mouse CD3e</td>
 <td>BD Biosciences</td>
 <td>553057</td>
 <td></td>
 </tr>
 <tr>
 <td>Purified NA/LE Hamster Anti-Mouse CD28</td>
 <td>BD Biosciences</td>
 <td>553294</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse IL-6 Recombinant Protein</td>
 <td>eBioscience</td>
 <td>14-8061-62</td>
 <td></td>
 </tr>
 <tr>
 <td>TGFbeta</td>
 <td>R&amp;D Systems</td>
 <td>240-B-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan blue solution 0.4 %</td>
 <td>Sigma-Aldrich</td>
 <td>66H2364</td>
 <td></td>
 </tr>
 <tr>
 <td>Pac Blue Rat Anti-Mouse CD4</td>
 <td>BD Biosciences</td>
 <td>558107</td>
 <td></td>
 </tr>
 <tr>
 <td>APC Rat Anti-Mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>553035</td>
 <td></td>
 </tr>
 <tr>
 <td>PE Conjugated Anti-mouse CD25</td>
 <td>eBioscience</td>
 <td>E01155-516</td>
 <td></td>
 </tr>
 <tr>
 <td>Alexa Fluor 700 Rat Anti-mouse IL-17</td>
 <td>BD Biosciences</td>
 <td>560820</td>
 <td></td>
 </tr>
 <tr>
 <td>Intracellular Cytokine Staining Starter Kit-Mouse</td>
 <td>BD Biosciences</td>
 <td>51-2041AK (559311)</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS CD4+CD25+ regulatory T cell isolation kit, mouse</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td></td>
 </tr>
 <tr>
 <td>ABAM </td>
 <td>Cellgro</td>
 <td>30-004-CI</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Corning, Cellgro</td>
 <td>10-040-CM</td>
 <td></td>
 </tr>
 <tr>
 <td>B 2-MercaptoEthanol</td>
 <td>MP Biomedical</td>
 <td>194834</td>
 <td>Hazardous</td>
 </tr>
 <tr>
 <td>Phorbol 12-Myristate 13-Acetate (PMA)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Calcium Salt</td>
 <td>Sigma-Aldrich</td>
 <td>13909-1ML</td>
 <td>Hazardous</td>
 </tr>
 <tr>
 <td>Brefeldin A (BFA)</td>
 <td>MP Biomedicals</td>
 <td>159027</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Capture mAb </td>
 <td>BD Biosciences</td>
 <td>555068</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Detection mAb </td>
 <td>BD Biosciences</td>
 <td>555067</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Standard</td>
 <td>eBioscience</td>
 <td>14-8171-80</td>
 <td></td>
 </tr>
 <tr>
 <td>IL-2 ELISA Kit</td>
 <td>BD Biosciences</td>
 <td>555148</td>
 <td></td>
 </tr>
 <tr>
 <td>TMB Substrate Reagent Set </td>
 <td>BD Biosciences</td>
 <td>555214</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>autoMACS Pro Cell Separator</td>
 <td>Miltenyi Biotec</td>
 <td>130-092-545</td>
 <td></td>
 </tr>
 <tr>
 <td>Sorvall Legend RT+ Centrifuge</td>
 <td>ThermoScientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Napco series 8000 WJ CO2 Incubator</td>
 <td>ThermoScientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>PTC-200 Peltier Thermal Cycler</td>
 <td>Biorad</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation and th17 differentiation of na&iuml;ve cd4 t lymphocytes

الخلايا TH17 هي مجموعة فرعية متميزة من الخلايا T التي تم العثور عليها لإنتاج انترلوكين 17 (IL-17)، وتختلف في وظيفة عن غيرها من مجموعات فرعية الخلايا التائية بما في ذلك الاستجابة Th1، الاستجابة Th2، والخلايا التائية التنظيمية. ظهرت الخلايا TH17 باعتباره الجاني المركزية في الاستجابات المناعية الالتهابية المرتبطة مماحكة مع العديد من اضطرابات المناعة الذاتية. في هذه الطريقة نحن تنقية الخلايا اللمفية تي من الطحال والغدد الليمفاوية من C57BL / 6 الفئران، وتحفيز خلايا CD4 + T المنقى تحت السيطرة والبيئات TH17 الذي يحفز. وتشمل البيئة الذي يحفز TH17 التحفيز في وجود مكافحة CD3 ومكافحة CD28 الأجسام المضادة، IL-6، وTGF-β. بعد حضانة لمدة 72 ساعة على الأقل ومدة تصل إلى خمسة أيام عند 37 درجة مئوية، ويتم تحليل الخلايا بعد ذلك لديها القدرة على إنتاج IL-17 من خلال التدفق الخلوي، QPCR، وELISAs. TH17 متباينة CD4 + CD25-T الخلايا يمكن استخدامها لمزيد من إلقاء الضوء على الدور الذي تلعبه الخلايا TH17 في ظهور وتطور المناعة الذاتية والمضيف ديقمم تهدف. علاوة على ذلك، يمكن TH17 التفريق بين CD4 + CD25 الخلايا اللمفية من نماذج متميزة خروج المغلوب / مرض الفئران تسهم في فهمنا للمصير الخلية اللدونة.

هذا البروتوكول التمايز TH17 يبدأ مع إزالة الطحال والإبطين، العضدية، المساريقي، عنق الرحم، والغدد الليمفاوية الأربية. تمثيل من المواقع من كل يمكن العثور عليها في الشكلين 2 و 3. الأرقام 1 و 5 على حد سواء توفر تمثيل مرئي من الأساليب المذكورة في ه?...

Watch this Scientific Journal Video about Isolation and Th17 Differentiation of NaÃƒÂ¯ve CD4 T Lymphocytes at JoVE.com

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

مع وظائف المستجيب متميزة عن غيرها فرعية الخلايا التائية والخلايا TH17 قد تورطت مركزيا في المناعة الذاتية للالتهابات. يوفر هذا البروتوكول في المختبر TH17 التمايز وسيلة لتحديد ما إذا كان من السذاجة اللمفاويات CD4 + T يمكن أن تفرق في خلايا TH17، ومواصلة دراسة دورها في المناعة الذاتية والاستجابة المضيف.

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 ...

isolation-and-th17-differentiation-of-nave-cd4-t-lymphocytes

Research

JoVE Journal

Immunology and Infection

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

34.3K Views.  The University of Florida.  مع وظائف المستجيب متميزة عن غيرها فرعية الخلايا التائية والخلايا TH17 قد تورطت مركزيا في المناعة الذاتية للالتهابات. يوفر هذا البروتوكول في المختبر TH17 التمايز وسيلة لتحديد ما إذا كان من السذاجة اللمفاويات CD4 + T يمكن أن تفرق في خلايا TH17، ...

Video: Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

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 ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

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

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

توجه التمييز بين الخلايا الجذعية المحفزة المستحثة نحو الخلايا اللمفية تي

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

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

عزل الخلايا اللمفاوية من المسالك التناسلية الماوس الغشاء المخاطي

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

تنبيغ الفيروسة الغدانية من CD4 الخلايا التائية ساذج لدراسة التمايز Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

الماوس السذاجة CD4<sup&gt; +</sup&gt; عزل T خلية و<em&gt; في المختبر</em&gt; التمايز إلى T مجموعات فرعية خلية

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

بروتوكول عزل فعال للاللمفاوية البائية والتائية من بلاطي اللوزتين الإنسان

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

التصوير CD4 T خلية الخلالية الهجرة في الأدمة الملتهبة

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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 ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

التمايز في المختبر من خلايا CD4 الإنسان + FOXP3 + المستحثة تنظيم الخلايا التائية (iTregs) من خلايا CD4 + T السذاجة باستخدام بروتوكول تحتوي على TGF-β-