<p class="jove_content"&gt; والكتاب أود أن أشكر جنيفر غريبة وبومان جريج على المساعدة التي قدموها مع تحليل التدفق الخلوي والفرز. وأيد هذا العمل من قبل عدد المنح المعاهد الوطنية للصحة 2P20 RR020171 من NCRR وجامعة للأموال بدء التشغيل كنتاكي إلى وزير الخارجية؛ GIE يقر بدعم من زمالة دراسات عليا الرئاسي والزمالة الفرصة كنتاكي.</p

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+Generation+and+Enhanced+Suppression+of+Human+CD4+CD25++Regulatory+T+Cells+by+Retinoic+Acid.">De Novo Generation and Enhanced Suppression of Human CD4+CD25+ Regulatory T Cells by Retinoic Acid.</a> Journal of Immunology. 183, 4119-4126 (2009).</li><li>Hori, S., Nomura, T., Sakaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+regulatory+T+cell+development+by+the+transcription+factor+Foxp3.">Control of regulatory T cell development by the transcription factor Foxp3.</a> Science (New York, N.Y.). 299, 1057-1061 (2003).</li><li>McHugh, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)CD25(+)+immunoregulatory+T+cells:+gene+expression+analysis+reveals+a+functional+role+for+the+glucocorticoid-induced+TNF+receptor.">CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.</a> Immunity. 16, 311-323 (2002).</li><li>Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+Self-Tolerance+Maintained+by+Cd25+Cd4+Regulatory+T+Cells+Constitutively+Expressing+Cytotoxic+T+Lymphocyte-Associated+Antigen+4.">Immunologic Self-Tolerance Maintained by Cd25+Cd4+Regulatory T Cells Constitutively Expressing Cytotoxic T Lymphocyte-Associated Antigen 4.</a> The Journal of Experimental Medicine. 192, 303-310 (2000).</li><li>Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD127+expression+inversely+correlates+with+FoxP3+and+suppressive+function+of+human+CD4++T+reg+cells.">CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells.</a> The Journal of Experimental Medicine. 203, 1701-1711 (2006).</li><li>Schraven, B., Kalinke, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD28+superagonists:+what+makes+the+difference+in+humans.">CD28 superagonists: what makes the difference in humans.</a> Immunity. 28, 591-595 (2008).</li><li>Reneer, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripherally+induced+human+Regulatory+T+cells+uncouple+Kv1.3+activation+from+TCR-associated+signaling.">Peripherally induced human Regulatory T cells uncouple Kv1.3 activation from TCR-associated signaling.</a> European Journal of Immunology. , (2011).</li></ol>

الكتاب ليس لديهم ما يكشف.

في حين نقل Treg يحمل وعدا العلاجية الهائلة في مجال مكافحة الكسب غير المشروع الرفض المناعة الذاتية وغيرها من الاضطرابات المناعية أو التهابات بوساطة، وأساليب لجيلهم كفاءة وصيانة مستقر حتى الآن لم يتم تطويرها. كما 1-5٪ فقط من تعميم خلايا تي هي Tregs، توسعها للرقابة والمفاضل?...

<table border="1" style=";text-align:right;direction:rtl"><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> Lymphoprep </td><td> محور الدرع </td><td> 1114547 </td><td> تبقى في درجة حرارة الغرفة </td></tr><tr><td> تبريد مقاعد البدلاء أعلى جهاز طرد مركزي </td><td> إيبندورف </td><td> 5810R </td><td></td></tr><tr><td> مشرق سطر عدادة الكريات </td><td> Hausser العلمية </td><td> 3110 </td><td></td></tr><tr><td> CD4 + T EasySep الإنسان كيت إثراء خلية </td><td> وقف سيل تكنولوجيز </td><td> 19052 </td><td></td></tr><tr><td> CD4 EasySep ذاكرة الإنسان + T كيت إثراء خلية </td><td> وقف سيل تكنولوجيز </td><td> 19157 </td><td></td></tr><tr><td> CD4 EasySep السذاجة الإنسان + T كيت إثراء خلية </td><td> وقف سيل تكنولوجيز </td><td> 19155 </td><td></td></tr><tr><td> من السهل الكبير EasySep ماجصاف </td><td> وقف سيل تكنولوجيز </td><td> 18001 </td><td> فضة </td></tr><tr><td> 5 مل البوليستيرين القاع جولة أنابيب </td><td> دينار بحريني الصقر </td><td> 352008 </td><td></td></tr><tr><td> 15 جهاز طرد مركزي أنابيب البولي بروبيلين مل </td><td> VWR </td><td> 89004-368 </td><td></td></tr><tr><td> 14 مل من مادة البولي بروبيلين المستديرة القاع أنابيب </td><td> دينار بحريني الصقر </td><td> 352059 </td><td></td></tr><tr><td> 50 جهاز طرد مركزي أنابيب البولي بروبيلين مل </td><td> VWR </td><td> 89004-364 </td><td></td></tr><tr><td> الإنسان لمكافحة الأجسام المضادة CD3 </td><td> الحيوي X خلية </td><td> BE0001-2 </td><td> استنساخ: OKT3 </td></tr><tr><td> 24 البوليستيرين خلية حسنا لوحة الثقافة </td><td> دينار بحريني الصقر </td><td> 353047 </td><td></td></tr><tr><td> 96 نسيج القاع جولة حسنا لوحة الثقافة </td><td> جرينير بيو واحد </td><td> 650180 </td><td></td></tr><tr><td> RPMI 1640 مع Glutamax-I والواق HEPES </td><td> Gibco </td><td> 72400 </td><td></td></tr> &lt;TR&gt; <td> الجنين المصل البقري (FBS) </td><td> Gibco </td><td> 16000 </td><td></td></tr><tr><td> β-المركابتويثانول </td><td> سيغما </td><td> M7522 </td><td></td></tr><tr><td> المؤتلف الإنسان TGF-β1 </td><td> eBioscience </td><td> 14-8348-62 </td><td></td></tr><tr><td> المؤتلف الإنسان IL-2 </td><td> eBioscience </td><td> 14-8029-63 </td><td></td></tr><tr><td> البقري ألبومين المصل (BSA) </td><td> النائب Biomedicals المؤتمر الوطني العراقي. </td><td> 810531 </td><td></td></tr><tr><td> 0.5 متر EDTA </td><td> Amresco </td><td> E177 </td><td></td></tr><tr><td> الماوس المضادة للالإنسان CD25-PE </td><td> Miltenyi بيوتيك </td><td> 130-091-024 </td><td> استنساخ: 4E3 </td></tr><tr><td> الماوس المضادة للالإنسان CD45RA-PE-Cy5 </td><td> eBioscience </td><td> 15-0458-42 </td><td> استنساخ: HI100 </td></tr><tr><td> الماوس المضادة للالإنسان CD127-APC </td><td> Miltenyi بيوتيك </td><td> 130-094-890 </td><td> استنساخ: MB15-18C9 </td></tr><tr><td> الدناز الثاني </td><td> Mف Biomedicals المؤتمر الوطني العراقي. </td><td> 190370 </td><td></td></tr><tr><td> MoFlo تدفق عداد الكريات </td><td> بيكمان كولتر </td><td></td><td></td></tr><tr><td> FlowJo برامج </td><td> شجرة نجمة، وشركة </td><td></td><td></td></tr><tr><td> برامج الخلية كويست برو </td><td> العلوم البيولوجية دينار بحريني </td><td></td><td></td></tr><tr><td> الوليد العجل المصل </td><td> Gibco </td><td> 16010 </td><td></td></tr><tr><td> L-الجلوتامين </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> AIM-V </td><td> Gibco </td><td> 0870112 </td><td></td></tr><tr><td> CellTrace تكاثر الخلايا CFSE كيت </td><td> إينفيتروجن </td><td> C34554 </td><td></td></tr><tr><td> Treg الخرز المفتش قمع </td><td> Miltenyi بيوتيك </td><td> 130-092-909 </td><td></td></tr><tr><td> البنسلين الستربتوميسين </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 40 خلية نايلون ميكرومتر الانفعالإيه </td><td> دينار بحريني الصقر </td><td> 352340 </td><td></td></tr></table>

generation of induced regulatory t cells from primary human na&iuml;ve and memory t cells

تطوير وصيانة CD4 + التنظيمية مناعة الخلايا التائية (Tregs) المساهمة في تحمل الطرفية اللازمة للبقاء في التوازن المناعية مع الكم الهائل من مستضدات الذات وcommensal في وعلى الجسم البشري. يمكن أن الاضطرابات في التوازن بين Tregs والتهابات الخلايا التائية التقليدية يؤدي في الباثولوجيا المناعية أو السرطان. على الرغم من أن ثبت الحقن العلاجية للTregs أن تكون فعالة في الفئران نماذج من التهاب القولون 1، النوع الأول من مرض السكري 2، والتهاب المفاصل الروماتيزمي والكسب غير المشروع مقابل المضيف المرض، و 4 عدة فروق جوهرية في مجال البيولوجيا Treg الإنسان مقابل 5 الماوس وبذلك حالت دون حتى استخدام السريري. استلزم عدم توافر العدد الكافي، والنقاء، والاستقرار، وخصوصية صاروخ موجه من Tregs العلاجية منصة حيوية للتنمية البشرية على Treg التي لتحسين الظروف لتوسعها المجراة سابقا 6. مد هنا نحنescribe وسيلة للتمايز Tregs التي يسببها (iTregs) من نفس واحدة الإنسان المانحة الدموية المحيطية والتي يمكن تقسيمها إلى أربع مراحل: عزل الخلايا الدموية المحيطية وحيدات النوى، واختيار المغناطيسي للخلايا CD4 + T، في خلية ثقافة المختبر، ومضان تنشيط الفرز الخلية (نظام مراقبة الأصول الميدانية) من مجموعات فرعية الخلايا التائية. منذ توقيع نسخ Treg عامل forkhead P3 مربع (FoxP3) هو عامل النسخ التنشيط التي يسببها البشر (7) ويجب عدم استخدام أي علامة أخرى فريدة من نوعها موجودا، لجنة توافقية من علامات لتحديد الخلايا التائية مع النشاط المكثف. بعد ستة أيام في الثقافة، لا يمكن ترسيم الخلايا في نظامنا إلى الخلايا التائية السذاجة، وخلايا تي الذاكرة أو iTregs على أساس التعبير النسبية من CD25 و CD45RA. كما الخلايا التائية الذاكرة والسذاجة لديها متطلبات مختلفة عن الاستقطاب وplasticities 8، قبل فرز السكان تي الخلية الأولية إلى + CD45RA وCD45RO + يمكن فرعية باستخدام البريد لدراسة هذه التناقضات. بما يتفق مع الآخرين، لدينا CD25 مرحبا CD45RA - iTregs مستويات عالية من التعبير عن FoxP3 9، GITR وCTLA 4-11 وانخفاض مستويات CD127 12. بعد FACS من كل السكان، ويمكن استخدام الخلايا الناتجة في فحص القامع الذي يقيم القدرة النسبية لتأخير انتشار carboxyfluorescein succinimidyl استر (CFSE) المسمى ذاتي الخلايا التائية.

Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃ¯ve and Memory T Cells at JoVE.com

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

ونحن تصف طريقة لتوليد خلايا تي التنظيمية، والذاكرة وساذجة من متبرع الدم البشري واحد. ويمكن مقارنة Tregs الاستقطاب ثم إلى مجموعات فرعية أخرى في مجموعة متنوعة من التطبيقات الوراثية والوظيفية مع تجانس الجينية، بما في ذلك فحص قمع بالتفصيل هنا أيضا.

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 ...

generation-induced-regulatory-t-cells-from-primary-human-nave-memory

Research

JoVE Journal

Immunology and Infection

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

23.9K Views.  University of Kentucky. ونحن تصف طريقة لتوليد خلايا تي التنظيمية، والذاكرة وساذجة من متبرع الدم البشري واحد. ويمكن مقارنة Tregs الاستقطاب ثم إلى مجموعات فرعية أخرى في مجموعة متنوعة من التطبيقات الوراثية والوظيفية مع تجانس الجينية، بما في ذلك فحص قمع بالتفصيل هنا أيضا.

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac تعديل النظام الأساسي للخلايا ينقول T الإنسان

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

أدوات جديدة لتوسيع الخلايا التائية التنظيمية من الأفراد HIV-1-المصابة

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

توليد خلايا T الإنسان مستضد خيفي محددة من الدم المحيطي

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

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

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

T خلايا التقاط البكتيريا التي كتبها Transinfection من الخلايا الجذعية

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

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

المظهرية والتحليل الوظيفي من المنشط خلايا تنظيم تي المعزولة من الفئران المصابة بفيروس المزمنة الليمفاوي المشيمي

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

تطوير المستمدة الخلية الجذعية خلايا T التنظيمية مستضد محددة ضد المناعة الذاتية

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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.

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

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 ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

تقييم الواجهة متشابك لخلايا تي البشرية الأولية من الدم والأنسجة اللمفاوية

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.