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.

이 비디오 간행물 및 프로토콜, 모든 동물의 절차는 주립 기관 바젤 슈타 트, 스위스에서 승인 한 동물 프로토콜에 따라 수행 하였다. 무균 조건 가능한 모든 절차를 수행합니다. 수술 1. 준비 <ol><li> 모든 수술 도구를 압력솥 사용하기 전에 거즈. </li><li> 따뜻한 패드를 가열 테이블 (재료 / 방법의 테이블)에 수술기구를 구성 할 수 있습니다. </li><li> 손가락 스트립 붕대를 엽니 다. (가제가 완전히 포함되어 있는지 확인) 면봉으로 상처 패드에 바셀린을 적용합니다. </li><li> 쥐를 달아 진통제 프레 놀핀에게 0.05 ㎎ / ㎏ 체중을 관리 할 수​​ 있습니다. 참고 :이 즉시 이식 후 진통을 제공 할 것입니다. </li></ol> 이식의 꼬리 피부의 2. 준비 <ol><li> CO 2 질식 및 D 당 기증자 마우스 (예를 들어, IA의 BM12)를 안락사절단하기 전에 소독 용액으로 꼬리를 isinfect. </li><li> 깨끗한 플라스틱 보드에 검은 줄무늬 아래로 꼬리를 놓고 중간 라인에 걸쳐 길이 방향으로 잘라, 단지 피부 층을 포함하는 표면 절개. </li><li> 그립 집게를 사용하여 꼬리의 피부를 당겨. </li><li> 10 ㎖ HBSS로 가득 10cm의 세포 배양 접시에 꼬리 피부를 넣습니다. </li></ol> 3. 수술 <ol><li> 투명 유도 상자 3 % 이소 플루오 란 용액 흡입받는 마우스 (예, C57BL / 6)을 마취. 참고 : 마우스가 수술 비행기 마취에 도달하는 것은 3 ~ 5 분입니다. 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. </li><li> 따뜻한 패드에 마우스를 올려 놓고 발가락이 곤란하여 각 동물의 반사 입 마스크와 엉덩이를 통해 1.5 % 이소 플루오 란을 적용합니다. </li><li> 받는 사람 생쥐의 등쪽 사이트 (예를 들어, C57BL / 6) 면도 마른 거즈 SW를 사용하여 오염 머리카락을 제거AB. </li><li> 무균 용액으로 면도 이식 사이트를 소독. </li><li> 배부의 오른쪽 / 중앙 사이트의 집게로 피부를 조이고 약 1 × 0.6 cm의 원형 절개를 만들기 위해 곡선 가위를 사용합니다. </li><li> 출혈이 발생하는 경우, 멸균 면봉으로 상처를 청소. </li><li> 절개의 크기를 평가하고 메스를 사용하여 꼬리 피부에서 예상 크기의 이식을 잘라. </li><li> 이식을 반올림 모서리를 낸다. </li><li> 멸균 면봉에 메스, HBSS의 건조 초과에 이식을로드하고받는 사람 마우스의 이식 침대에 배치합니다. </li><li> 기증자와 호스트 피부의 중복을 방지하고 독점적으로 접촉 영역에서 조직 접착제를 적용합니다. </li><li> 마우스 허리 주위에 주름없이 석고를 적용합니다. </li><li> 마우스에 석고를 해결하기 위해 접착제가 아닌 탄력 붕대를 적용 (3 MM은 이동). </li><li> 붕대의 상단 복부 사이트의 3mm에 대한 가위로 절개. 주 :이 TR을 피하기위한 것이다복구 단계에서 마우스의 치아 apping. </li><li> 마취 입 마스크를 제거하고 깨어 때까지 가열 패드에 이식 된 쥐를 유지합니다. </li><li> 완전히 복구 된 경우에만 자신의 새장에 이식 된 쥐를 반환하고 붕대가 너무 꽉되지 않도록 자신의 이동성을 평가합니다. </li><li> 다음 수술을 진행하기 전에 70 % 에탄올로 모든 수술기구를 소독. </li></ol> 4. 수술 후 케어 <ol><li> 식수 (4 ㎎ / ㎖)로 해열 함유 시럽을 추가, 7 일 동안을 관리 할 수​​ 있습니다. </li><li> 이 복구되면 동물의 방에 마우스를 반환합니다. </li><li> 밀접하게 붕대 장소 (1 주)에 시간을 우울증이나 다른 행동 변화 (식사 또는 부동), 비정상적인 모양 (손질의 부족) 또는 자세 (pilo 직립 또는 새우등 자세) 등의 임상 증상에 마우스를 모니터링 할 수 있습니다. </li></ol> 5. 붕대 제거 (6 ~ 7 일 후 이식) <ol><li> 마취투명 유도 상자 (위 참조)의 3 % 이소 플루오 란 용액을 흡입 thetize 마우스. </li><li> 동맥 가위 붕대와 석고를 잘라 조심스럽게 제거합니다. 주 : 이식 사이트의 스트레칭 마십시오. </li><li> 임상 증상에 대한 이식 생쥐 (4.3 참조) 모니터 및 피부 이식 (표 1)의 거부에 대한 점수. 주 : 이식 점수 시스템 등의 피부에 비해 꼬리 피부에 다른 머리 질감을 활용합니다. 이식과 거부의 모양은 3 등급으로 분류됩니다. C57BL / 6 마우스의 면역 동종 피부 (MHC II 일치하지 않는, 예를 들어, IA BM12) 이식은 일반적으로 12~15일 포스트 이식 (자신의 데이터와 15 ~ 20)에서 거부됩니다. </li></ol>

<ol>
	<li>Mahillo, B., Carmona, M., &#193;lvarez, M., White, S., Noel, L., Matesanz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Data+in+Organ+Donation+and+Transplantation.">Global Data in Organ Donation and Transplantation.</a> transplantation. 92 (10), 1069-1074 (2011).</li><li>Halleck, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+of+immunosuppression.">New perspectives of immunosuppression.</a> Transplantation proceedings. 45 (3), 1224-1231 (2013).</li><li>Wood, K. J., Bushell, A., Jones, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+unresponsiveness+to+alloantigen+in+vivo.+a+role+for+regulatory+T+cells.">Immunologic unresponsiveness to alloantigen in vivo. a role for regulatory T cells.</a> Immunological reviews. 241 (1), 119-132 (2011).</li><li>Sykes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+tolerance:+mechanisms+and+application+in+clinical+transplantation.">Immune tolerance: mechanisms and application in clinical transplantation.</a> Journal of Internal Medicine. 262 (3), 288-310 (2007).</li><li>McFarland, H. I., Rosenberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+allograft+rejection..">Skin allograft rejection..</a> Current protocols in immunology. , (2009).</li><li>McKenzie, I. F., Morgan, G. M., Sandrin, M. S., Michaelides, M. M., Melvold, R. W., Kohn, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B6.C-H-2bm12.+A+new+H-2+mutation+in+the+I+region+in+the+mouse.">B6.C-H-2bm12. A new H-2 mutation in the I region in the mouse.</a> The Journal of experimental medicine. 150 (6), 1323-1338 (1979).</li><li>McIntyre, K. R., Seidman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+of+mutant+I-A+beta+bm12+gene+is+evidence+for+genetic+exchange+between+mouse+immune+response+genes.">Nucleotide sequence of mutant I-A beta bm12 gene is evidence for genetic exchange between mouse immune response genes.</a> Nature. 308 (5959), 551-553 (1984).</li><li>Stuart, P. M., Beck-Maier, B., Melvold, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Provocation+of+skin+graft+rejection+across+murine+class+II+differences+by+non--bone-marrow-derived+cells.">Provocation of skin graft rejection across murine class II differences by non--bone-marrow-derived cells.</a> Transplantation. 37 (4), 393-396 (1984).</li><li>Hausmann, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+through+a+motif+in+the+alphabeta+T+cell+receptor.">Positive selection through a motif in the alphabeta T cell receptor.</a> Science. 281 (5378), 835-838 (1998).</li><li>Bill, J., Ronchese, F., Germain, R. N., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+mutant+amino+acids+to+alloantigenicity.">The contribution of mutant amino acids to alloantigenicity.</a> The Journal of experimental medicine. 170 (3), (1989).</li><li>Monaco, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression+and+tolerance+for+clinical+organ+allografts.">Immunosuppression and tolerance for clinical organ allografts.</a> Current Opinion in Immunology. 1 (6), 1174-1177 (1989).</li><li>Rosenberg, A. S., Singer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+basis+of+skin+allograft+rejection:+an+in+vivo+model+of+immune-mediated+tissue+destruction.">Cellular basis of skin allograft rejection: an in vivo model of immune-mediated tissue destruction.</a> Annual Review of Immunology. 10, 333-358 (1992).</li><li>Kingsley, C. I., Nadig, S. N., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+tolerance:+lessons+from+experimental+rodent+models.">Transplantation tolerance: lessons from experimental rodent models.</a> Transplant international : official journal of the European Society for Organ Transplantation. 20 (10), 828-841 (2007).</li><li>Larsen, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+acceptance+of+skin+and+cardiac+allografts+after+blocking+CD40+and+CD28+pathways.">Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways.</a> Nature. 381 (6581), 434-438 (1996).</li><li>Tomura, M., Nakatani, I., Murachi, M., Tai, X. G., Toyo-oka, K., Fujiwara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+allograft+responses+induced+by+interleukin-6,+which+selectively+modulates+interferon-gamma+but+not+interleukin-2+production.">Suppression of allograft responses induced by interleukin-6, which selectively modulates interferon-gamma but not interleukin-2 production.</a> Transplantation. 64 (5), 757-763 (1997).</li><li>Ring, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-gamma+is+necessary+for+initiating+the+acute+rejection+of+major+histocompatibility+complex+class+II-disparate+skin+allografts.">Interferon-gamma is necessary for initiating the acute rejection of major histocompatibility complex class II-disparate skin allografts.</a> Transplantation. 67 (10), 1362-1365 (1999).</li><li>Rosenberg, A. S., Finbloom, D. S., Maniero, T. G., Vander Meide, P. H., Singer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+prolongation+of+MHC+class+II+disparate+skin+allografts+by+in+vivo+administration+of+anti-IFN-gamma+monoclonal+antibody.">Specific prolongation of MHC class II disparate skin allografts by in vivo administration of anti-IFN-gamma monoclonal antibody.</a> Journal of immunology. 144 (12), 4648-4650 (1950).</li><li>Goes, N., Sims, T., Urmson, J., Vincent, D., Ramassar, V., Halloran, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbed+MHC+regulation+in+the+IFN-gamma+knockout+mouse.+Evidence+for+three+states+of+MHC+expression+with+distinct+roles+for+IFN-gamma.">Disturbed MHC regulation in the IFN-gamma knockout mouse. Evidence for three states of MHC expression with distinct roles for IFN-gamma.</a> Journal of immunology. 155 (10), 4559-4566 (1995).</li><li>Surquin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-4+deficiency+prevents+eosinophilic+rejection+and+uncovers+a+role+for+neutrophils+in+the+rejection+of+MHC+class+II+disparate+skin+grafts.">IL-4 deficiency prevents eosinophilic rejection and uncovers a role for neutrophils in the rejection of MHC class II disparate skin grafts.</a> Transplantation. 80 (10), 1485-1492 (2005).</li><li>Gaylo, A. E., Laux, K. S., Batzel, E. J., Berg, M. E., Field, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+rejection+of+MHC+class+II-disparate+skin+allografts+in+mice+treated+with+farnesyltransferase+inhibitors.">Delayed rejection of MHC class II-disparate skin allografts in mice treated with farnesyltransferase inhibitors.</a> Transplant immunology. 20 (3), 163-170 (2009).</li><li>Bose, A., Inoue, Y., Kokko, K. E., Lakkis, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+perforin+down-regulates+CD4+and+CD8+T+cell-mediated+immune+responses+to+a+transplanted+organ.">Cutting edge: perforin down-regulates CD4 and CD8 T cell-mediated immune responses to a transplanted organ.</a> Journal of immunology. 170 (4), 1611-1614 (2003).</li></ol>

The authors declare that they have no competing financial interests.

피부 이식은 거부하고 T 세포에 따라 허용 공부를위한 널리 사용되는 방법입니다. 피부 이식 모델이 확립 된 이후, 여러 가지 적응 및 변경 사항이 적용되었습니다. 설명 과정에서 IA BM12 꼬리 피부 이식은 마취 가스 (이소 플루오 란)를 사용하여 수행됩니다. 마취 가스의 사용은 이식 된 생쥐에 응력을 감소 실행 및 생쥐 복구 시간을 감소시킨다. 절차는 귀 또는 등쪽 피부 조직보다 말리는 ...

이러한 피부, 심장, 신장 등 고형 장기의 이식은 지금 전 세계적으로 의료 행위 1의 표준 절차입니다. 성공적으로 장기 이식은 기증자의 주요 조직 적합성 항원을 인식받는 사람의 면역 시스템의 활성화에 의해 거부 될 수 있습니다. 따라서 이식 환자는 면역 억제 약물이 치료가 필요합니다. 생쥐 동종 피부 이식은 1955 년에 메다와 동료에 의해 설립 이후 주요 조직 적합성 복합체 (MHC) 클래스 I 및 II로 기술 대상 분자를 식별하는 데 도움이이었다. 그 이후로, 피부 이식 모델은 연속적으로 수정 및 T 세포 서브 세트의 역할 및 이식 거부 2-4을 억제하는 화학적 및 생물학적 개입의 관련성을 연구하도록 채택되었다. 귀 트렁크에서 피부는 준비하기가 더 어렵습니다 꼬리 피부 5보다 저산소증 및 괴사에 더 민감하다; 그러나,이식 절차는 비슷합니다. 또한 꼬리의 피부 이식의 모니터링으로 인해 피부의 특징적인 머리 짜임새에 용이합니다. 이 문서는 CD4 + T 세포 - 매개 동종 이식 거부 반응 및 생쥐의 허용 오차의 다양한 측면의 연구를 허용 MHC 클래스 II 불일치 꼬리 피부 이식에 대한 자세한 절차를 제공합니다. (IA BM12라고 함) MHC 클래스 II 분자 IA B의 자연 3 점 돌연변이는 6-9 C57BL / 6 마우스 8 피부 동종 이식의 거부 반응을 유발하기에 충분하다. IA의 BM12 분자는 다양한 αβ-T 세포 수용체 Vα2Vβ8-TCR 특정 T 세포의 TCR-트랜스 제닉 마우스 (10)를 생성하기 위해 식별 된 구비 C57BL / 6 생쥐 (TCR) 체인과 CD4 + T 세포를 활성화한다. Vα2Vβ8-TCR 특정 T 세포의 대체 송금 면역 결핍 C57B에서 거부 모델을 수립하는 데 사용되어왔다L / 6 Rag2 - / - IA BM12의 피부에 이식 된 쥐. 기증자와받는 사람 사이의 유전 적 차이는 이식 수용과 거부의 결과에 영향을 미칠. 이식의 다른 유형이있다 :자가 이식받는 사람의 개인 자체에서 이식이다; syngrafts 및 동종 이식은 각각 유 전적으로 동일하고 유 전적으로 관련이없는 개인의 이식입니다. 다른 동종 장기 이식의 수용 환자와 마우스 모델 11,3,4의 화학 및 생물 학적 개입에 의해 증명되었다. 기본적인 접근 방식에서, 항 CD3 항체 처리 C57BL / 6 마우스는 IA BM12 꼬리 피부 (게시되지 않은 데이터)의 장기 생존율을 보였다. 세포받는 생쥐에 이식하기 전에 CD4 +와 CD8 + T의 고갈은 MHC 클래스 I 및 II 일치하지 않는 이식 (12 목사)의 수용의 결과. 흥미롭게도, 피부 이식의 거부 반응은 CD4 +의 존재에 따라 달라집니다 </sup&gt; T 세포 (12 계). 이 모델에서, 규제 T 세포와 항체 억제와 공동 자극 분자를 차단하여 다른 면역 세포 사이의 특정한 상호 작용을 대상으로하는 것은 허용 (게시되지 않은 데이터)를 유도 할 수 있습니다. 실제로, CD40 및 CD28을 모두 차단하는 것은 장기적으로 피부 이식 허용 오차 13, 14되었다. 꼬리 피부 이식은 다른 장기의 이식에 비해 수행하기 쉬운 모니터링하기 쉽습니다. 또한, 꼬리, 피부 이식을 준비하기 쉽고 다른 피부 조직에 비해 국소 빈혈에 덜 민감하다. 주입 마취제 달리, 이식 동안 마취 가스 (이소 플루오 란)의 사용 절차와 수신자 복구 시간을 모두 단축시킨다. 불완전 상처 치유 및 이식 거부로 이어질 수 꼬리 피부 이식의 컬링은, 조직 접착제의인가에 의해 방지된다. 또한, IA BM12 꼬리 피부 이식 모델은 독점적으로 CD4를 활성화 <sup&gt; + (동일한 유전 적 배경) 면역 및 면역 결핍 마우스 모두에서 T 세포는 결과의 해석을 용이. 이 프로토콜은 화학 및 생물 학적 개입을 할 수있는, 신뢰할 수있는 재생 가능한 쉽게 모니터링 마우스 모델에 대해 설명합니다. 이 모델은 테일 피부 이식의 거부와 관용 유도를 조사하기위한 것입니다.

<table border="0" cellpadding="0" cellspacing="0" width="831">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hansaplast Finger Strips</td>
			<td>Beierdorf AG</td>
			<td style="width:119px;">REF.76861</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile gauze, 5 x 5cm, 8ply</td>
			<td>MaiMed GmbH</td>
			<td>21010</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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.

첫 번째 방법에서 C57BL / 6 마우스는 IA의 BM12 동종 이식 및 IA B 형 syngrafts 이식되었다. 붕대를 제거한 후, C57BL / 6 마우스 (그림 1A)의 접촉 영역의 폐쇄하지 않고 상처 치유의 명백한 징후를 접목. 붕대를 제거한 후, CD4 + T 세포 매개 염증, 괴사 영역의 모양 (붉은 반점)과 이식 (그림 1B-C, 못 기호) 13 이후 일 이내에 C57BL / 6 마우스의 IA의 BM12 동종</su...

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.

마우스에 동종 CD4 T 세포 반응을 연구하기 위해 테일 피부 이식

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 조회수.  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.

비디오: 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 특정 T 세포 생성 Allogeneic 조혈 줄기 세포 이식 후 바이러스 감염을 예방 / 치료

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

연구

면역학 및 감염병학

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.

Diabetogenic CD4의 입양 전송하여 마우스의 가속 제 1 형 당뇨병 유도 + T 세포

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.

나이브 CD4의 아데노 바이러스 형질은 Treg의 분화를 연구하는 세포를 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.

분리 및 나이브 CD4 T 림프구의 TH17 분화

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.

이펙터 CD4의 입양 전송하여 쥐과 장내 염증의 유도<sup&gt; +</sup&gt; CD45RB<sup&gt; 높은</sup&gt; 면역 결핍 마우스에게 T 세포

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.

마우스 나이브 CD4<sup&gt; +</sup&gt; T 세포의 분리 및<em&gt; 시험관</em&gt; T 세포 하위 집합으로 차별화

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.

염증이 진피의 이미징 CD4 T 세포 간질 마이그레이션

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.

생체 외에서 그리고 Vivo에서 T, B 및 골수성 세포 진압 활동 및 이식 받는 체액 응답의 평가

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.

인간 T 세포 회수 응답에서 Vivo에서 공부 하는 흡입 물집 프로토콜

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.

감염 된 Ifnar1/- 생쥐의 Immunoprivileged 장기에서 Zika 바이러스 관련 T 세포 응답의 평가

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