فإن الكتاب أود أن أشكر جميع أعضاء المختبر رينولدز في روزاليند فرانكلين جامعة الطب والعلوم، والمختبر تشن دونغ في جامعة تكساس مركز أندرسون للسرطان لتعظيم الاستفادة من هذا البروتوكول. وأيد هذا العمل من خلال منحة لJMR من المعاهد الوطنية للصحة (K22AI104941).

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الكتاب تعلن أي المصالح المالية المتنافسة.

في حين أن الطحال يحتوي على خلايا ث الساذجة، فإن نسبة هذه الفئة من السكان في الغدد الليمفاوية هو أعلى من ذلك بكثير. والفشل في تحديد بشكل صحيح وإزالة العقد اللمفاوية في هذا البروتوكول يؤدي إلى العائد الضعيف للخلايا ساذجة. وهذا يمكن أن يكون من الصعب وخصوصا في الفئران الأ...

مفهوم الأنساب متميزة أو مجموعات فرعية من CD4 + T المساعد وكانت (ث) خلايا موجودة منذ الجزء الأخير من القرن التاسع 20 1. الاعتراف مستضد وما شابه ذلك بحضور النتائج إشارات costimulatory في عدة جولات من انتشار الخلوي والتمايز في نهاية المطاف إلى المستجيب ث الخلايا. نوع ث الخلايا المولدة خلال هذه العملية يتوقف على البيئة خلوى الحالية خلال تفعيل 2. في البداية، كان يعتقد خلايا ث ساذجة لاستقطاب في 2 الأنساب المتميزة التالية مستقبلات الخلايا التائية (TCR) التنشيط، costimulatory CD28 ربط، وخلوى الإشارات. وتتميز نوع 1 الخلايا المساعدة (TH1) من خلال إنتاج المستجيب لها من خلوى IFNγ فضلا عن احتياجاتهما لIL-12 الإشارات خلال 3،4 عملية التمايز. في نهاية المطاف تم اكتشاف أن خلايا TH1 متباينة لديها الصورة الجينية التي تتميز معظم متميز بذ التعبير عن مربع T عامل النسخ الأسرة، Tbx21 (T-رهان)، والتي تعتبر المنظم الرئيسي للبرنامج جيني TH1 5. وعلاوة على ذلك، يمكن أن IL-12 وكذلك IFNγ تعزيز T-رهان التعبير 6،7. في الاستجابة المناعية، وخلايا TH1 مهمة للدفاع المضيف ضد مسببات الأمراض داخل الخلايا وكذلك المروجين قوي للالتهاب المناعة الذاتية. في المقابل، خلايا من النوع 2 المساعد (TH2) تتطلب IL-4 لتنميتها والسيتوكينات المستجيب، بما في ذلك IL-4 و IL-5، وIL-13، هي مهمة للقيادة استجابات الخلايا B وهي المسببة للأمراض في الحساسية 8، 9. وعلى غرار TH1 الخلايا، تم العثور على خلايا TH2 للتعبير عن نفسه منظم النسخي سيدهم، ووصف GATA-3 10،11. ومن المثير للاهتمام، فإن وجود السيتوكينات استقطاب وتوليد نسب ث المحددة هي معادية لتطوير الآخرين 2،12، مما يوحي بأن مجموعة فرعية فقط ث معينة قد تصبح المهيمنة خلال إعادة المناعياستجابته. منذ التعرف على Th1 و TH2 الأنساب، وقد أثبت المزيد من العمل حتى أكثر فريدة من نوعها مجموعات فرعية من الخلايا التائية المساعدة، بما في ذلك مسامي المساعد (TFH)، IL-9-إنتاج (Th9)، وIL-22-إنتاج (Th22) (مؤخرا استعراضها في 13). لأغراض التجارب في المختبر التمايز، وهذا البروتوكول تركز فقط على اثنين من مجموعات فرعية ث إضافية، ووصف خلايا T التنظيمية (Treg) وIL-17-إنتاج خلايا CD4 + T (Th17). CD25 + خلايا T التنظيمية يمكن أن يحدث بشكل طبيعي (nTreg) في الغدة الصعترية. ويمكن أيضا أن يتسبب خلايا ث ساذجة (iTreg) لتصبح التنظيمية في محيط (استعراضها في 14،15). كلا النوعين من Tregs تعبر عن عامل النسخ مميزة، يطلق مربع forkhead P3 (Foxp3)، وهو أمر حاسم لآليات قمع المستجيب لها والتي تشمل إنتاج قابل للذوبان المضادة للالتهابات الوسيط، IL-2 الاستهلاك، وآليات الخلية التي تعتمد على الاتصال 14،15. عدم وجود Foxp3 النتائج في التعبير الشديد، الأعضاء المتعددة اضطراب المناعة الذاتية وصفته ديسريغولاتيون المناعي، polyendocrinopathy، الأمعاء، ومتلازمة X-مرتبط (IPEX)، مما يدل على الدور الحاسم لهذا فرعية ث في حل التهاب وتنظيم التسامح الطرفية في تقرير المصير مستضدات 16. في المختبر، الخلايا المساعدة CD4 + T ساذجة يصل تنظيم Foxp3 وأصبح ملتزما برنامج Treg على التحفيز مع IL-2 و TGF-β 14،15. قد يكون هناك معتدلة إلى اللدونة كبيرة في CD4 + T الأنساب الخلية، وخاصة عند النظر في إنتاج السيتوكينات الوحيد (استعراضها في 17،18). ومع ذلك، لأغراض في المختبر بروتوكولات التمايز، وسوف نناقش كل مجموعة فرعية كما نسب فريدة من نوعها. مؤخرا، تم تحديد مجموعة فرعية من خلايا Th17 التي تنتج خلوى IL-17 كما نسب فريدة من نوعها مع وظائف الموالية للالتهابات التي هي المسببة للأمراض وخاصة أثناء التهاب المناعة الذاتية 19-21. خلايا Th17 تعبر عن عامل النسخ فريدة من نوعها، ووصف المتعلقة ريتينويد اليتيم مستقبلات غاما ر (RORγt) التي تنسق برنامج جيني Th17 22. TGFβ المهم لتوليد Th17 النسب من خلال تحريض RORγt. ومع ذلك، يعتقد أن تأثير TGFβ إشارات للحث على الالتزام فقط Th17 على التآزر مع IL-6 (استعراضها في 12). وقد أظهرت دراسات أخرى أن مجموعة متنوعة من الإشارات الأخرى التي يمكن أن تنظم بشكل إيجابي التزام Th17، بما في ذلك IL-1β، وزيادة الصوديوم، وTLR اشارة 23-26. وأشارت تقارير أخرى إلى أن خلايا Th17 المسببة للأمراض في الجسم الحي هي تلك التي تجاوز الواقع TGFβ الإشارات وبدلا من ذلك تعتمد على مزيج من IL-1، IL-6، وIL-23 للتمايز على 27. وبالتالي، قد تكون مشتقة خلايا Th17 من مجموعة متنوعة من مسارات الإشارات. لأغراض هذا البروتوكول، وشائعة الاستخدام (TGFβ وIL-6) مسارا لTh17 التزام النسبوستعرض. البروتوكولات التمايز هو موضح أدناه لجميع الأنساب المستجيب تعتمد على الأجسام المضادة ثابت كما المحفزات لTCR وCD28 في جميع أنحاء كامل التجربة. ومع ذلك، فقد أظهرت آخرون أن تفعيل TCR مع الخلايا العارضة للمستضد 28 أو مكافحة CD3 وأضداد CD28 مع الهامستر الأجسام المضادة ربط عبر لمدة 2 أيام 29 هي أيضا وسيلة فعالة للغاية من تحريض جيل من مختلف مجموعات فرعية ث. بروتوكول المعروضة هنا يبني على أساليب ذكرت سابقا لعزل خلايا CD4 + T الفئران من الأجهزة اللمفاوية الثانوية 30 و توليد خلايا Th17 31. احد الفرق الرئيسي هو أن هذا البروتوكول يعتمد على استخدام فارز خلية لعزل خلايا CD4 + T ساذجة من الأنسجة اللمفاوية. ومع ذلك، العديد من الشركات تقدم الآن مجموعات فصل السريعة التي يمكن أن تثري للخلايا CD4 + T السذاجة، والتي قد تكون قادرة على تجاوز شرط وأو الفرز اعتمادا على التجربة. الطرق والكواشف الواردة في هذا البروتوكول هي التي نستخدمها بشكل روتيني وتجد أن تكون الأكثر فعالية. ومع ذلك، نضع في اعتبارنا أن الكواشف ومنهجيات بديلة موجودة لكثير من الخطوات الواردة أدناه، والأمر متروك إلى المختبر الفردية لتحديد ما سوف تعمل بشكل أفضل لتحقيق أغراضهم.

<table><tbody><tr><td>Complete RPMI:</td><td></td><td></td><td>Warm in a 37 &lt;sup&gt;o&lt;/sup&gt;C water bath before use</td></tr><tr><td>RPMI 1640 Media</td><td>Life Technologies</td><td>11875119</td><td></td></tr><tr><td>10 % FBS</td><td>Life Technologies</td><td>26140-079</td><td></td></tr><tr><td>1000X 2-mercaptoethanol</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>100X Pen/Strep</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>100X L-glutamine</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>120 micron nylon mesh</td><td>Amazon</td><td>CMN-0120-10YD</td><td>Cut into 2 cm&lt;sup&gt;2&lt;/sup&gt; squares and autoclave</td></tr><tr><td>Alternative: 100 micron cell strainers</td><td>Fisher</td><td>08-771-19</td><td>Alternative to cutting nylon mesh</td></tr><tr><td>autoMACS running buffer</td><td>Miltenyi</td><td>130-091-221</td><td>Warm in a 37 &lt;sup&gt;o&lt;/sup&gt;C water bath before use</td></tr><tr><td>autoMACS rinsing solution</td><td>Miltenyi</td><td>130-091-222</td><td>Warm in a 37 &lt;sup&gt;o&lt;/sup&gt;C water bath before use</td></tr><tr><td>CD4 beads</td><td>Miltenyi</td><td>130-049-201</td><td></td></tr><tr><td>ACK lysis buffer</td><td>Life Technologies</td><td>A10492-01</td><td></td></tr><tr><td>Cytokines:</td><td></td><td></td><td></td></tr><tr><td>Human (h) IL-2</td><td>Peprotech</td><td>200-02</td><td></td></tr><tr><td>Recombinant mouse (rm) IL-4</td><td>Peprotech</td><td>214-14</td><td></td></tr><tr><td>rmIL-6</td><td>R &amp;amp; D Systems</td><td>406-ML-025</td><td></td></tr><tr><td>rmIL-12</td><td>Peprotech</td><td>210-12</td><td></td></tr><tr><td>hTGFb</td><td>R &amp;amp; D Systems</td><td>240-B-010</td><td></td></tr><tr><td>Antibodies:</td><td></td><td></td><td></td></tr><tr><td>2C11 (anti-CD3)</td><td>BioXcell</td><td>BE0001-1</td><td></td></tr><tr><td>37.51 (anti-CD28)</td><td>BioXcell</td><td>BE0015-1</td><td></td></tr><tr><td>11B11 (anti-IL-4)</td><td>BioXcell</td><td>BE0045</td><td></td></tr><tr><td>XMG1.2 (anti-IFNg)</td><td>BioXcell</td><td>BE0055</td><td></td></tr><tr><td>anti-CD62L-FITC</td><td>BioLegend</td><td>104406</td><td>Use at 1:100</td></tr><tr><td>anti-CD25-PE</td><td>BioLegend</td><td>102008</td><td>Use at 1:400</td></tr><tr><td>anti-CD4-PerCP</td><td>BioLegend</td><td>100434</td><td>Use at 1:1000</td></tr><tr><td>anti-CD44-APC</td><td>BioLegend</td><td>103012</td><td>Use at 1:500</td></tr><tr><td>Phorbol&amp;nbsp; 12-myristate 13 acetate (PMA)</td><td>Sigma-Aldrich</td><td>P-8139</td><td>Prepare a stock at 0.1 mg/ml in DMSO and freeze aliquots at -20 &lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>I-0634</td><td>Prepare a stock at 0.5 mg/ml in DMSO and freeze aliquots at -20 &lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Brefeldin A</td><td>eBioscience</td><td>00-4506-51</td><td>Use at 1:1000</td></tr></tbody></table>

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.

النقطة الوقت لتحليل التمايز يمكن أن تختلف تبعا لحالة ث يجري اختبارها فضلا عن قوة T تنشيط مستقبلات الخلية. بعد 2-3 أيام من التمايز، والخلايا يمكن تصور بواسطة المجهر الضوئي لتحديد مدى انتشار الخلايا T. سوف الآبار واظهار انتشار واسع والتثاقل من خلايا من المرجح أن تكون جاه?...

Watch this Scientific Journal Video about Mouse NaÃƒÂ¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets at JoVE.com

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 ...

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JoVE Journal

Immunology and Infection

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

51.9K Views.  Rosalind Franklin University of Medicine and Science. Naïve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of naïve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals. 

Video: Mouse Naïve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

جيل من الخلايا المستحثة تي التنظيمية من المرحلة الابتدائية الإنسان خلايا T ساذجة والذاكرة

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

اشتقاق خلايا T<em&gt; في المختبر</em&gt; من الفأر الجنينية الخلايا الجذعية

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

إعداد CD4<sup&gt; +</sup&gt; خلايا T للتحليل GD3 وGD2 غانغليوزيد غشاء التعبير بواسطة الميكروسكوب

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

فيروسات تنبيغ من الخلايا التائية المساعدة كنهج الوراثي لدراسة آليات السيطرة على التمايز وظيفة

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

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-

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

نموذج ماوس في فيفو إلى تدبير السذاجة CD4 تي خلية التنشيط وانتشارها والتمايز Th1 الناجمة عن الخلايا الجذعية المشتقة من نخاع العظام

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

<meta charset="utf8"></head><body>تمايز الخلايا Th17 من الخلايا التائية CD4 + الساذجة باستخدام قياس التدفق الخلوي

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

<meta charset="utf8"></head><body>تسليط الضوء على المؤلف: تحقيق تمايز عالي النقاء في المختبر لخلايا Th17 باستخدام تعديل تركيز السيتوكين

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

التمايز في المختبر للخلايا التائية CD4 + الساذجة إلى خلايا Th17 المسببة للأمراض في الماوس

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

الماوس السذاجة CD4

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جيل من الخلايا المستحثة تي التنظيمية من المرحلة الابتدائية الإنسان خلايا T ساذجة والذاكرة

تنبيغ الفيروسة الغدانية من CD4 الخلايا التائية ساذج لدراسة التمايز Treg

العزلة والتمايز TH17 من السذاجة CD4 T الخلايا اللمفاوية

اشتقاق خلايا T

إعداد CD4

فيروسات تنبيغ من الخلايا التائية المساعدة كنهج الوراثي لدراسة آليات السيطرة على التمايز وظيفة

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

نموذج ماوس في فيفو إلى تدبير السذاجة CD4 تي خلية التنشيط وانتشارها والتمايز Th1 الناجمة عن الخلايا الجذعية المشتقة من نخاع العظام

التمايز في المختبر للخلايا التائية CD4 ⁺ الساذجة إلى خلايا Th17 المسببة للأمراض في الماوس

التمايز في المختبر للخلايا التائية CD4 ⁺ الساذجة إلى خلايا Th17 المسببة للأمراض في الماوس