This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R. We thank E. Palmer and B.T.H. Hausmann for mice and technical expertise.

في هذا المنشور الفيديو والبروتوكول، وأجريت كافة الإجراءات الحيوانية وفقا لبروتوكول الحيوانية التي وافقت عليها السلطة كانتون بازل شتات، سويسرا. تنفيذ كافة الإجراءات في ظروف معقمة حيثما كان ذلك ممكنا. 1. إعداد جراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الأوتوكلاف جميع الأدوات الجراحية والشاش قبل الاستخدام. </li><li style=";text-align:right;direction:rtl"> تسخين سادة دافئة وتنظيم الأدوات الجراحية على الطاولة (جدول المواد / طرق). </li><li style=";text-align:right;direction:rtl"> فتح الضمادات الشريط الاصبع. تطبيق الفازلين على لوحة الجرح مع مسحة القطن (تأكد من تغطية الشاش تماما). </li><li style=";text-align:right;direction:rtl"> وزن الفئران وإدارة تسكين البوبرينورفين 0.05 مغ / كغ من وزن الجسم. ملاحظة: هذا سيوفر تسكين مباشرة بعد الزرع. </li></ol> 2. إعداد الذيل الجلد لزرع <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الموت ببطء الفئران المانحة (على سبيل المثال، IA bm12) في CO 2 الخنق ودisinfect ذيول بمحلول تطهير قبل الختان. </li><li style=";text-align:right;direction:rtl"> وضع الذيل مع شريط أسود أسفل على لوحة بلاستيكية نظيفة وقطع عليه طوليا في جميع أنحاء خط الوسط، مما يجعل شق سطحية تنطوي فقط على طبقة الجلد. </li><li style=";text-align:right;direction:rtl"> سحب قبالة الجلد من الذيل باستخدام ملقط تجتاح. </li><li style=";text-align:right;direction:rtl"> وضع الذيل الجلد في صحن 10 سم ثقافة الخلية مليئة 10 مل HBSS. </li></ol> 3. الإجراءات الجراحية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير الفئران المتلقي (على سبيل المثال، C57BL / 6) عن طريق استنشاق حل isofluorane 3٪ في صندوق الاستقراء شفافة. ملاحظة: يستغرق 3-5 دقيقة للفأر للوصول إلى الطائرة التخدير الجراحي. استخدام مرهم الطبيب البيطري على العينين لمنع جفاف بينما تحت التخدير. </li><li style=";text-align:right;direction:rtl"> وضع الماوس على لوحة دافئة وتطبيق 1.5٪ isofluorane عبر قناع الفم وتقويم كل العاكسة الحيوانية أخمص القدمين معسر. </li><li style=";text-align:right;direction:rtl"> يحلق موقع الظهري من الفئران المتلقي (على سبيل المثال، C57BL / 6) وإزالة الشعر تلويث باستخدام الشاش جنوب غرب الجافةمن أساسها. </li><li style=";text-align:right;direction:rtl"> تطهير حليق موقع الزرع مع الحل العقيم. </li><li style=";text-align:right;direction:rtl"> قرصة الجلد بالملقط في موقع الوسط / اليمين للظهر واستخدام مقص منحني لجعل شق جولة من حوالي 1 × 0.6 سم. </li><li style=";text-align:right;direction:rtl"> في حالة حدوث نزيف، ثم تنظيف الجرح مع مسحة القطن المعقم. </li><li style=";text-align:right;direction:rtl"> تقييم حجم شق وقطع الزرع من الحجم المقدر من الذيل الجلد باستخدام المشرط. </li><li style=";text-align:right;direction:rtl"> تقليم زوايا لتقريب عملية الزرع. </li><li style=";text-align:right;direction:rtl"> تحميل زرع على مشرط، الزائدة الجافة من HBSS على مسحة القطن المعقم ووضعه في السرير الكسب غير المشروع من الماوس المتلقي. </li><li style=";text-align:right;direction:rtl"> تجنب تداخل المانحة والمضيفة الجلد والأنسجة وتطبيق لاصق على منطقة الاتصال حصرا. </li><li style=";text-align:right;direction:rtl"> تطبيق الجص دون الطويات حول الخصر الماوس. </li><li style=";text-align:right;direction:rtl"> تطبيق لاصقة ضمادة غير مرنة لإصلاح الجص إلى الماوس (3 ملم تحول). </li><li style=";text-align:right;direction:rtl"> شق مع مقص حوالي 3 ملم في الموقع بطني العليا للضمادة. ملاحظة: هذا هو لتجنب آرapping الأسنان الماوس، أثناء مرحلة الانتعاش. </li><li style=";text-align:right;direction:rtl"> إزالة مخدر الفم قناع والحفاظ على الفئران المزروع على لوحة التسخين حتى مستيقظا. </li><li style=";text-align:right;direction:rtl"> العودة الفئران المزروعة إلى أقفاصها فقط عندما تعافى تماما وتقييم قدرتهم على التنقل لضمان أن ضمادة ليست ضيقة جدا. </li><li style=";text-align:right;direction:rtl"> تعقيم جميع الأدوات الجراحية مع الايثانول 70٪ قبل الشروع في عملية جراحية المقبل. </li></ol> 4. رعاية ما بعد الجراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة شراب المحتوية على الباراسيتامول لمياه الشرب (4 ملغ / مل) وادارته لمدة 7 أيام. </li><li style=";text-align:right;direction:rtl"> العودة إلى غرفة الماوس الحيوان بمجرد استعادتها. </li><li style=";text-align:right;direction:rtl"> عن كثب مراقبة الفئران لعلامات سريرية بما في ذلك الاكتئاب أو التغيرات السلوكية الأخرى (الأكل أو الجمود)، ومظهر غير طبيعي (عدم وجود الاستمالة) أو الموقف (بيلو-الانتصاب أو الموقف منحنية) للمرة الضمادة في مكانها (1 أسبوع). </li></ol> 5. إزالة ضمادة (6-7 أيام بعد الزرع) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> انيسthetize الفئران عن طريق استنشاق حل Isofluorane 3٪ في صندوق الاستقراء شفافة (انظر أعلاه). </li><li style=";text-align:right;direction:rtl"> قطع ضمادة والجص مع مقص الشريان وبعناية إزالتها. ملاحظة: تجنب تمتد من موقع الزرع. </li><li style=";text-align:right;direction:rtl"> مراقبة الفئران لزرع علامات سريرية (انظر 4.3) ويسجل لرفض ترقيع الجلد (الجدول 1). ملاحظة: نظام نقاط الكسب غير المشروع يستفيد من القوام الشعر المختلفة على الجلد الذيل مقارنة الظهرية الجلد. يصنف مظهر عملية الزرع، ورفضه في 3 درجات. وعادة ما يتم رفض الجلد خيفي (MHC II متطابقة، على سبيل المثال، IA bm12) الطعوم من C57BL / 6 الفئران مناعيا في غضون 12-15 يوما زرع آخر (البيانات الخاصة و 15-20). </li></ol>

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The authors declare that they have no competing financial interests.

زرع الجلد هو طريقة تستخدم على نطاق واسع لدراسة الرفض والتسامح تعتمد على خلايا تي. منذ تأسيس نموذج زرع الجلد، وطبقت العديد من التعديلات والتغييرات. في الإجراء وصفها، يتم تنفيذ IA bm12 زرع الذيل الجلد باستخدام الغاز مخدر (isofluorane). استخدام التخدير الغاز يقلل من وقت ال?...

زرع الأعضاء الصلبة مثل الجلد والقلب والكلى هو الآن إجراء القياسية في الممارسة الطبية في جميع أنحاء العالم 1. يمكن رفض الأعضاء المزروعة بنجاح من قبل تفعيل نظام المناعة المتلقي، الذي يعترف مستضدات التوافق النسيجي الرئيسي من الجهة المانحة. مرضى زرع بالتالي تحتاج إلى علاج بالأدوية المثبطة للمناعة 2. تأسست خيفي زرع الجلد في الفئران عن طريق مدور وزملاؤه في عام 1955، وكان مفيدا لتحديد الجزيئات المستهدفة وصفها لاحقا معقدة التوافق النسيجي الرئيسي (MHC) الصف الأول والثاني. منذ ذلك الحين، تم زرع الجلد نموذج تعديل بشكل مستمر وتكييفها لدراسة دور مجموعات فرعية الخلايا T وأهمية الكيميائية والبيولوجية التدخل في قمع رفض الكسب غير المشروع 2-4. الجلد من الأذن والجذع هي أكثر صعوبة لإعداد وهم اكثر عرضة لنقص الأكسجة ونخر الجلد من الذيل 5؛ ومع ذلك،الإجراء هو زرع مماثلة. بالإضافة رصد الذيل زرع الجلد من السهل نظرا لنسيج الشعر سمة من الجلد. توفر هذه المقالة إجراء مفصلة لفئة MHC II عدم تطابق زرع الذيل الجلد الذي يسمح لدراسة جوانب مختلفة من CD4 + T الخلية بوساطة رفض طعم خيفي والتسامح في الفئران. الطفرة ثلاث نقاط الطبيعي في MHC الدرجة الثانية IA جزيء ب (وتسمى IA bm12) 6-9 يكفي للحث على رفض المغايرة الجلد في C57BL / 6 الفئران 8. جزيء bm12 IA ينشط خلايا CD4 + T مع مختلف αβ-T الخلايا مستقبلات (TCR) سلاسل من C57BL / 6 الفئران، التي تم تحديدها بين الخلايا T-Vα2Vβ8 TCR محددة من أجل توليد ماوس TCR-10 المعدلة وراثيا. وقد استخدم نقل بالتبني من الخلايا T-Vα2Vβ8 TCR محددة لوضع نموذج الرفض في C57B العوز المناعيL / 6 Rag2 - / - الفئران زرعها مع IA bm12 الجلد. الاختلافات الجينية بين المتبرع والمتلقي تؤثر على نتائج القبول والرفض زرع. هناك أنواع مختلفة من عمليات زرع: زرع طعم ذاتي هي من الفرد المتلقي نفسه؛ syngrafts والمغايرة هي من زرع الأفراد متطابقة وراثيا وجينيا لا علاقة لها على التوالي. وقد ثبت قبول مختلفة خيفي زرع الأعضاء من خلال التدخل الكيميائية والبيولوجية في المرضى ونماذج الماوس 11،3،4. في النهج الأساسي، أظهرت مكافحة CD3 المعالجة الضد C57BL / 6 الفئران البقاء على قيد الحياة لفترة طويلة من IA bm12 الذيل الجلد (بيانات غير منشورة). أدى استنزاف CD4 + و CD8 + T الخلايا قبل زرع في الفئران المتلقية في قبول MHC من الدرجة الأولى والثانية ترقيع غير متطابقة (القس في 12). ومن المثير للاهتمام، ورفض ترقيع الجلد يعتمد على وجود CD4 + </sup&gt; خلايا T (القس في 12). في هذا النموذج، تستهدف تفاعلات معينة بين الخلايا المناعية المختلفة من خلال منع الجزيئات costimulatory مع الأجسام المضادة أو قمع مع الخلايا التائية التنظيمية قد تحفز التسامح (بيانات غير منشورة). في الواقع، ومنع كل من CD40 CD28 وأدى إلى طويلة الأجل طعم خيفي الجلد التسامح 13،14. زرع الذيل الجلد هو سهلة لتنفيذ وسهلة لرصد مقارنة زرع الأعضاء الأخرى. بالإضافة إلى ذلك، زرع الذيل الجلد هي سهلة التحضير وأقل عرضة لنقص التروية من أنسجة الجلد الأخرى. وعلى النقيض من حقن التخدير، واستخدام الغاز مخدر (isofluorane) خلال زرع يقصر كل من الإجراء والانتعاش مرة المستفيدة. الشباك من زرع الذيل الجلد، والتي قد تؤدي إلى عدم اكتمال التئام الجروح ورفض الكسب غير المشروع، ومنعه من خلال تطبيق لاصق الأنسجة. علاوة على ذلك، IA bm12 الذيل الجلد نموذج زرع ينشط حصرا CD4 <sup&gt; + الخلايا التائية في كل مناعيا والفئران العوز المناعي (من نفس الخلفية الوراثية) تسهيل تفسير النتائج. يصف هذا البروتوكول نموذج الفأر موثوق بها، واستنساخه رصدها بسهولة أن يسمح للتدخل الكيميائية والبيولوجية. ويهدف نموذج للتحقيق في الرفض والتسامح تحريض زرع الذيل الجلد.

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			<td>Fine iris scissor&#160;</td>
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			<td>Tissue forceps</td>
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			<td style="width:119px;">3006</td>
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			<td>disposable</td>
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			<td height="21" style="height:21px;width:299px;">Vaseline</td>
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transplantation of tail skin to study allogeneic cd4 t cell responses in mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

في النهج الأول تم زرع C57BL / 6 الفئران مع IA المغايرة bm12 وIA ب syngrafts. بعد إزالة الضمادات، ترقيع واضح علامات التئام الجروح دون إغلاق منطقة الاتصال في C57BL / 6 الفئران (الشكل 1A). بعد إزالة الضمادات، أدى CD4 + T التهاب بوساطة الخلايا إلى ظهور مناطق نخرية (ا?...

Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

زرع الجلد الذيل لدراسة خيفي CD4 T الردود الخليوي في الفئران

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

transplantation-tail-skin-to-study-allogeneic-cd4-t-cell-responses

Research

JoVE Journal

Immunology and Infection

12.3K Views.  University of Basel and University Hospital Basel. Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Video: Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

توليد خلايا T - Multivirus محددة لمنع / علاج الالتهابات الفيروسية خيفي بعد زرع الخلايا الجذعية المكونة للدم

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

المعجل نوع 1 الحث السكري في الفئران عن طريق نقل بالتبني من CD4 + T خلايا سكري

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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 ...

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

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

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

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

العزلة والتمايز TH17 من السذاجة CD4 T الخلايا اللمفاوية

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

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

تحريض الفئران المعوية التهاب عن طريق نقل بالتبني من المستجيب CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; عالية</sup&gt; خلايا T في الفئران العوز المناعي

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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 ...

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 ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

في المختبر و في فيفو التقييم تي وب وخلايا النقوي النشاط القمعية واستجابات خلطيه من المتلقين زرع

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

بروتوكول نفطة شفط لدراسة الخلية تي البشرية استدعاء الاستجابات في فيفو

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

وصف الاستجابات ايستب الغلوبولين المناعي تعتمد على الغدة الصعترية، والغدة الصعترية مستقلة في الفئران باستخدام مقايسة الممتز المرتبط بالانزيم

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

زرع الجلد الذيل لدراسة خيفي CD4 T الردود الخليوي في الفئران

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