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.

Bu videoyu yayın ve protokolde, tüm hayvan prosedürleri Kanton Kurumu, Basel-Stadt, İsviçre tarafından onaylanan hayvan protokole uygun olarak yapılmıştır. Steril koşullarda mümkün tüm işlemleri gerçekleştirmek. Cerrahi 1. Hazırlanması <ol><li> Tüm cerrahi aletler otoklav ve kullanımdan önce pamuğu. </li><li> Sıcak ped ısıtın ve tablo (Malzeme / Yöntemleri Tablo) cerrahi aletler düzenlemek. </li><li> Parmak şerit bandaj açın. (Gazlı bez tamamen kapalı olduğundan emin olun) bir pamuklu çubukla yara pad üzerinde vazelin uygulayın. </li><li> Fareler tartılır ve analjezi buprenorfin 0.05 mg / kg vücut ağırlığı yönetmek. Not: Bu hemen nakli sonrası analjezi sağlayacaktır. </li></ol> Nakli için Kuyruk-cilt 2. Hazırlanması <ol><li> CO 2 boğulma ve d başına donör fareler (örneğin, IA bm12) Euthanizeeksizyon öncesi dezenfeksiyon solüsyonu ile kuyrukları isinfect. </li><li> Temiz bir plastik gemide siyah şerit aşağı ile kuyruk yerleştirin ve orta hattı boyunca uzunlamasına kesilmiş, sadece deri tabakasını içeren yüzeysel bir kesi yapmak. </li><li> Kavrama forseps kullanarak kuyruk derisini çekin. </li><li> 10 ml HBSS ile dolu bir 10 cm hücre kültürü çanağı içinde kuyruk-cilt koyun. </li></ol> 3.. Cerrahi Prosedür <ol><li> Şeffaf bir indüksiyon kutu içinde bir% 3 izofloran çözeltisinin inhalasyonu yolu ile alıcı fareler (örneğin, C57BL / 6) anestezisi. Not: fare cerrahi uçak anestezi ulaşmak için bu 3-5 dakika sürer. Anestezi altında iken kuruluğunu önlemek için gözleri veteriner merhem kullanın. </li><li> Sıcak pad üzerinde fare yerleştirin ve ayak kısma her hayvan refleks bir ağız maskesi ve eşek üzerinden% 1,5 isofluorane geçerlidir. </li><li> Alıcı farelerin dorsal sitesi (örneğin, C57BL / 6) tıraş ve kuru bir gazlı bez sw kullanarak kirlenmesine tüyleriab. </li><li> Aseptik çözelti ile tıraş transplantasyon bölgesine dezenfekte. </li><li> Dorsumun sağ / orta site forseps ile cilt sıkın ve yaklaşık 1 x 0.6 cm çapında yuvarlak kesi yapmak için kavisli makas kullanın. </li><li> Kanama oluşursa, o zaman steril pamuklu çubukla yarayı temizleyin. </li><li> Kesi boyutunu değerlendirmek ve neşter kullanarak kuyruk-cilt büyüklük tahmini bir nakli kesti. </li><li> Nakli yuvarlak köşeleri Trim. </li><li> Steril pamuklu çubukla üzerinde neşter, HBSS&#39;nin kuru aşan üzerinde nakli yükleyin ve alıcı fare greft yatağına yerleştirin. </li><li> Donör ve alıcı cilt çakışmasını önlemek ve sadece kontak zonda doku yapıştırıcısı uygulayın. </li><li> Fare beline kırışıklıklar olmadan bir sıva uygulanır. </li><li> Fare sıva düzeltmek için yapışkan olmayan elastik bandaj uygulanır (3 mm değiştirdi). </li><li> Bandajın üst ventral sitesinde 3 mm ile ilgili makasla kesin. Not: Bu tr kaçınmaktırkurtarma aşamasında fare diş aritalandırma. </li><li> Anestetik-ağız maskesi çıkarın ve uyanık kadar ısıtma yastığı nakledilen fareler tutun. </li><li> Tamamen iyileşti sadece kendi kafeslerine nakledilen fareler dönün ve bandaj çok sıkı olmadığından emin olmak için onların hareketliliği değerlendirmek. </li><li> Sonraki ameliyatın geçmeden önce% 70 etanol ile tüm cerrahi aletler sterilize. </li></ol> 4. Post-operatif Bakım <ol><li> Içme suyu (4 mg / ml) için parasetamol içeren şurubu ilave edin ve 7 gün boyunca uygulanması. </li><li> Kurtarılamaz kez hayvan odasına fare dönün. </li><li> Yakından bandaj yerde (1 hafta) olduğu zamandır depresyon veya diğer davranış değişiklikleri (yeme ya da hareketsizlik), anormal görünüm (tımar eksikliği) veya postürü (pilo-ereksiyon ya da kambur duruş) dahil olmak üzere klinik belirtileri için fare monitör. </li></ol> 5.. Bandaj Temizleme (6-7 Gün Post-transplantasyon) <ol><li> Anesşeffaf bir indüksiyon kutusu (yukarıya bakın) ve% 3 İzofloran çözeltisinin inhalasyonu yolu ile thetize farenin. </li><li> Arter makasla bandaj ve alçı kesme ve dikkatlice çıkarın. Not: transplant site germe kaçının. </li><li> Klinik bulguları nakledilen farelerin (4.3 bakınız) Monitör ve deri grefti (Tablo 1) reddi için puan. Not: Graft puan sistemi, dorsal cilde göre kuyruk-cilt üzerinde farklı saç dokular yararlanır. Nakli ve reddedilme görünüm 3. sınıflarda kategorilere ayrılır. C57BL / 6 imünokompetan farelerin allojenik deri (MHC II uyumsuz, örneğin, IA bm12) greftler genellikle 12-15 gün transplantasyon sonrası (kendi veri ve 15-20) içinde reddedilir. </li></ol>

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The authors declare that they have no competing financial interests.

Deri nakli reddi ve T hücrelerine bağımlı tolerans araştırılması için yaygın olarak kullanılan bir yöntemdir. Deri nakli modeli kurulduğundan beri, çeşitli uyarlamalar ve değişiklikler uygulandı. Açıklanan prosedürde, IA bm12 kuyruk deri nakli anestezi gazı (izofloran) kullanılarak gerçekleştirilir. Gaz anestezi kullanımı nakledilen fareler üzerindeki stres azalır yürütme ve fareler kurtarma, zamanını azaltır. Prosedür kulak veya dorsal cilt dokusunun daha kıvrılabilecek k...

Bu tür deri, kalp ve böbrekler gibi katı organ nakli artık dünya çapında tıbbi uygulamada 1 standart bir işlemdir. Başarıyla nakledilen organlar vericinin MHC antijenleri tanıyan alıcı bağışıklık sisteminin aktivasyonu ile reddedilebilir. Bu nedenle nakledilen hasta immünosüpresif ilaçlar 2 ile tedavi gerekir. Farelerde allojenik deri transplantasyonu 1955 yılında Medawar ve arkadaşları tarafından kurulan ve daha sonra MHC kompleksi (MHC) sınıf I ve II olarak tanımlanan hedeflenen moleküllerinin belirlenmesi için yararlı oldu. O zamandan beri, deri nakli modelinin sürekli değiştirilmiş ve T hücre alt rolü ve greft reddini 2-4 bastırmada kimyasal ve biyolojik müdahale ilişkisini incelemek için adapte edilmiştir. Kulak ve gövde cilt hazırlamak için daha zordur ve kuyruk-cilt 5 daha hipoksi ve nekroz daha yatkındır; Bununla birlikte,Transplantasyon prosedürü; benzer. Buna ek olarak kuyruk deri nakillerinin izlenmesi nedeniyle cildin karakteristik saç dokusu kolaydır. Bu makalede, CD4 + T hücre aracılı allograft reddi ve farelerde tolerans farklı yönlerini çalışmaya olanak MHC sınıf II uyumsuzluk tail-deri nakli için ayrıntılı bir işlem sağlar. (IA bm12 olarak da adlandırılır), MHC sınıf II molekülü IA b doğal üç nokta mutasyonu 6-9 C57BL / 6 fare deri 8 allograftlara neden olmak için yeterlidir. IA bm12 molekülü çeşitli αβ-T hücresi reseptörü Vα2Vβ8 TcR-spesifik T hücreleri, bir TcR transgenik fare 10 oluşturmak için tespit edilmiştir, bunlar arasında C57BL / 6 fareden (TcR) zincirleri ile CD4 + T hücreleri aktive eder. Vα2Vβ8-TCR-özgü T hücrelerinin adoptif transfer C57B bağışıklık yetersizliği olan bir ret model kurmak için kullanılmıştırL / 6 RAG2 - / - IA bm12 deri ile nakledilen fareler. Alıcı ve verici arasındaki genetik farklılıklar nakli kabul ve reddedilme sonucu etkileyebilir. Nakillerinin farklı türleri vardır: otogreftleri alıcı bireyin kendisinden nakli olan; syngrafts ve allogreftler sırasıyla genetik olarak özdeş ve akrabalık ilişkisi olmayan kişilerden nakilleri vardır. Farklı alojenik organ nakillerinin kabul hasta ve fare modellerinde 11,3,4 kimyasal ve biyolojik müdahale ile gösterilmiştir. Temel bir yaklaşım olarak, anti-CD3 antikoru ile tedavi edilen C57BL / 6 fareleri IA bm12 tail-derinin (yayınlanmamış veriler) uzun süre hayatta kalması gösterdi. Hücreleri alıcı farelerde transplantasyon öncesi CD4 + ve CD8 + T tükenmesi MHC sınıf I ve II uyumsuz greft (12 in rev.) kabul sonuçlandı. İlginç bir şekilde, deri graftları reddi CD4 + varlığına bağlıdır </sup&gt; T-hücreleri (12 Rev.) Bu modelde, düzenleyici T hücreleri ile antikor veya bastırılması ile birlikte uyaran molekülleri bloke farklı bağışıklık hücreleri arasındaki etkileşimleri hedef toleransı (yayınlanmamış veriler) neden olabilir. Gerçekten de, CD40 ve CD28, blokaj uzun süreli deri allograft toleransı 13,14 yol açtı. Kuyruk deri nakli, diğer organların nakli ile karşılaştırıldığında gerçekleştirmek kolay ve izlenmesi kolaydır. Buna ek olarak, kuyruk deri nakli hazırlanması kolaydır ve diğer deri dokularının çok iskemiye daha az duyarlıdır. Enjekte anestezik aksine, transplantasyon sırasında anestezik gaz (izofluoran) kullanımı prosedürü ve alıcı iyileşme süresini kısaltır hem de. Eksik yara iyileşmesi ve aşı reddi yol açabilir kuyruk deri nakli, kıvrılmaları, doku yapıştırıcı uygulanması ile önlenir. Ayrıca, IA bm12 kuyruk deri nakli modeli sadece CD4 aktive <sup&gt; + (Aynı genetik arka plan), imünokompetan ve bağışıklık yetersizliği olan farelerde hem de T hücrelerinin sonuçlarının yorumlanmasını kolaylaştırmak. Bu protokol, kimyasal ve biyolojik müdahale için izin veren güvenilir, tekrar üretilebilir ve kolay bir şekilde kontrol fare modeli tarif edilmektedir. Bu model, kuyruk deri nakillerinin reddedilmesi ve tolerans indüksiyonu araştırılması için tasarlanmıştır.

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			<td height="21" style="height:21px;width:299px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:231px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Betadine Standard solution</td>
			<td>Mundipharma</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton swab</td>
			<td style="width:183px;">Carl Roth GmbH</td>
			<td style="width:119px;">31025060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dafalgan , UPSA</td>
			<td>Bristol Myers Squibb SA</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Hansaplast Finger Strips</td>
			<td>Beierdorf AG</td>
			<td style="width:119px;">REF.76861</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Histoacryl Tissue adhesive</td>
			<td style="width:183px;">Braun</td>
			<td style="width:119px;">REF.1050052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leukotape classic , 2cm x 10 m</td>
			<td>BSN Medical SAS</td>
			<td>REF.02204-00</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">PBS, Phosphate-Buffered Saline, pH 7.4</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:119px;">10010015</td>
			<td>GIBCO</td>
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			<td height="21" style="height:21px;">Sterile gauze, 5 x 5cm, 8ply</td>
			<td>MaiMed GmbH</td>
			<td>21010</td>
			<td></td>
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			<td height="21" style="height:21px;">Surgical instruments&#160;</td>
			<td style="width:183px;">FST</td>
			<td style="width:119px;">11003-12</td>
			<td>Narrow pattern forceps,&#160;</td>
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			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14095-11</td>
			<td>Fine iris scissor curved,</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14094-11</td>
			<td>Fine iris scissor&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14010-15</td>
			<td>Mayo scissors,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14080-11</td>
			<td>Artery scissors ball tip 11.5 cm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">11021-14</td>
			<td>Tissue forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Surgical Blade No.20</td>
			<td style="width:183px;">Swann-Morton LTD</td>
			<td style="width:119px;">3006</td>
			<td>Carbon Steel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Surgical blade Handles</td>
			<td style="width:183px;">Swann-Morton LTD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe, 1ml</td>
			<td style="width:183px;">ARTSANA</td>
			<td style="width:119px;"></td>
			<td>disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temgesic , Buprenorphine</td>
			<td style="width:183px;">ESSEX Chemie AG</td>
			<td style="width:119px;"></td>
			<td>0.3mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Tissue Culture dishes 10 cm, 60.1 cm2</td>
			<td style="width:183px;">TPP</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Vaseline</td>
			<td style="width:183px;">Vifor SA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Warm pad</td>
			<td style="width:183px;">Solis</td>
			<td style="width:119px;"></td>
			<td>Type 223</td>
		</tr>
	</tbody>
</table>

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.

Bir birinci yaklaşımda, C57BL / 6 fareleri IA bm12 allogreft ve IA b syngrafts ile transplante edildi. Bandaj çıkarılmasından sonra C57BL / 6 fareler (Şekil 1A) &#39;de temas bölgesinin kapanmaması yara iyileşmesinin belirgin belirtileri greft. Bandaj çıkarılmasından sonra CD4 + T hücresi aracılı iltihaplanma, nekrotik alanlarında görünümü (kırmızı lekeler) ve transplantasyon (Şekil 1B-C, dolu semboller) sonra 13 gün içinde C5...

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.

Farelerde allojenik CD4 + T hücre Cevapları Eğitim için Kuyruk Cilt Nakli

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

12.2K Görüntüleme.  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.

Multivirus-spesifik T hücrelerinin üretilmesi Allojenik Hematopoetik Kök Hücre Nakli sonra Viral Enfeksiyonlar önleme / tedavi etmek için

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

İmmünoloji ve Enfeksiyon

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.

Diyabetojenik CD4 Kabul edilmiş Transfer ile Farelerde Hızlandırılmış Tip 1 Diyabet İndüksiyon + T Hücreleri

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.

Naif CD4 adenoviral İletimi Treg Farklılaşma Eğitim Hücreler T

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&amp;#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.

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

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.

Efektör CD4 Evlatlık transferi ile Kemirgen Bağırsak Enflamasyon indüksiyonu<sup&gt; +</sup&gt; CD45RB<sup&gt; Yüksek</sup&gt; Bağışık yetmezliği olan farelere içine T Hücreleri

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&amp;#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.

Fare Naif CD4<sup&gt; +</sup&gt; T Hücre İzolasyonu ve<em&gt; In vitro</em&gt; T hücre alt içine Farklılaşma

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.

Iltihaplı Dermisinde içinde görüntüleme CD4 + T hücre Geçiş Göç

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.

Vitro ve In Vivo değerlendirme T, B ve Myeloid hücrelerin süpresif etkinlik ve nakli alıcılardan gelen Humoral yanıt

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.

İnsan T-hücre hatırlama yanıt Vivo içinde çalışmaya emme Blister iletişim kuralı

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

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Virüslü Ifnar1- / - farelerin Immunoprivileged organlarında Zika virüs özgü T hücreli yanıt-e doğru değerlendirilmesi

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...