우리는 Drs에 감사드립니다. 도움이 덧글에 대한 로버트 BONNEAU 닐 크리스텐슨. 이 작품은 의학 자금의 펜실베니아 주립 대학에 의해 지원되었다.

1. 비장과 BDC2.5 쥐의 림프절에서 T 세포의 분리 <ol><li> diabetogenic CD4 + T 세포의 기증자로 6 주 된 사전 당뇨병 여성 BDC2.5 마우스를 사용합니다. 소변 포도당 측정 (아래 참조)에 의해 결정되는 마우스는 당뇨병이 없어야합니다. </li><li> CO 2 질식을 사용하여 각 마우스를 안락사 및 무균 조건 하에서 비장, 겨드랑이 및 상완 림프절을 제거합니다. 비장을 제거하려면, 70 % 에탄올로 모피를 흡수 한 다음 잘라 피부를 철회. 비장은 마우스의 왼쪽에 어두운 붉은 기관으로 볼 수 있습니다. 작은 가위를 사용하여 복막의 1 인치 절개를 부드럽게 작은 핀셋 한 쌍의 중앙에 비장을 파악. 조심스럽게 결합 조직과 연결된 지방 최대한 트림과 비장을 제거합니다. </li><li> 얼음에 15 ML 원뿔 튜브 10 ML Dulbecco&#39;s으로 수정 이글의 중간 (DMEM)에서 비장과 림프절을 수집합니다. </li><li> 일을 사용하여 단일 세포 현탁액을 준비합니다부드럽게 70 μm의 셀 스트레이너 (여과기 당 하나의 비장과 스트레이너 당 4-6 림프절) 같은 50 ML 원뿔 튜브로를 통해 림프 기관을 누릅니다 멸균 10 ML의 주사기 플런저의 전자 끝. </li></ol> 이 과정에서, 스트레이너에서 세포의 회복을 최대화하기 위해 1 ML DMEM 여러 번 각 스트레이너 린스. <ol start="5"><li> RT에서 12 분 동안 300 ~ 400 XG에 15 ML 원뿔 튜브와 원심 분리기에 50 ML 원뿔 튜브에서 수집 된 세포를 전송합니다. </li><li> 적혈구를 용해하는, 상층 액을 버리고, 5 ML 암모늄 - 염화 칼륨 (ACK) 버퍼 (산도 7.2)에서 세포를 다시 일시 중지하고 5 분 RT에서 ACK 세포 현탁액을 품어. </li><li> ACK 세포 현탁액에 DMEM 10 ML을 추가하고 위와 같이 튜브를 원심 분리기. 10 ML DMEM에 한 번 세포 펠렛을 씻으십시오. </li></ol> 2. Diabetogenic CD4의 형광 활성 세포 정렬 + BDC2.5 마우스에서 T 세포 <ol><li> 5 ML 외과의에서 다시 일시 중지합니다 세포버퍼를 함유 성 및 판 블루 염색 배제하여 가능한 세포 (위상차 현미경과 혈구를 사용하여)의 수를 계산합니다. </li><li> FACS 버퍼를 사용하여 5 × 10 7 세포 / ML에 세포 현탁액 볼륨을 조정합니다. ~ 제거 1 × 10 염색 제어 당 6 셀 (1 아무 얼룩, 3 단 얼룩). 비 활성화 된 유전자 변형 CD4 + 안티 CD4와 T 세포 (APC), 안티 TCR Vβ4 (FITC)와 15 ML 튜브에 반대로 CD62L (PE) 단일 클론 항체 (단클론 항체)에 대한 샘플을 얼룩. 어둠 속에서 4시에 20 ~ 30 분 동안 제조업체에서 권장하는 단클론 항체의 농도 ° C를 사용하여 세포 염색을 수행합니다. </li><li> &gt; 배 염색 반응 볼륨 FACS 버퍼와 샘플 및 단일 얼룩 컨트롤을 씻으십시오. (1-2 X 10 6 세포 / 샘플을 정렬 ML 300 μL 하나의 얼룩 컨트롤)과 얼음에 저장 다음까지 300 ~ 400 XG에서 튜브를 원심 분리, 상층 액을 제거하고 FACS 버퍼에 세포를 다시 일시 중지합니다 단계. </li><li> 35 μm의 세포를 통해 세포 샘플을 전달스트레나 캡 튜브는 세포 덩어리를 제거합니다. CD4에 대한 샘플을 정렬 + TCR Vβ4 + CD62L + 훈련 연산자 세포 분류기의 셀. 15 ML 튜브에 3 ML DMEM으로 정렬 세포를 수집합니다. 2 × 10 총 4 개 이벤트 / 초의 정렬 속도 1.5 X 10 8 셀 (3 공여 생쥐에서 얻은 세포의 대략적인 개수) 정렬하려면, 설치 등 2.5-3 시간을 허용합니다. 기대 ~ 2.5 × 10 6 비 활성화 된 유전자 변형 CD4 + 세포 분류 한 후 마우스 당 T 세포. </li><li> 정렬의 끝에 정렬 된 세포의 절대 수를 기록하고 300 ~ 400 X g에서 12 분을 원심 분리기. 뜨는을 취소하고 다시 중단 세포 (2 × 10 6 세포 / ㎖)의 멸균 인산 완충 입양 T 세포의 이동을위한 식염수 (PBS) (MG 2 + / 칼슘 2 + 무료). </li></ol> 3. BDC2.5 쥐에서 Diabetogenic CD4의 입양 전송 + T 세포 <ol><li> 다시 일시 중지합니다 FACS - 정제 된 CD4 부드럽게, 1 ML의 주사기와 18-1 ½ 게이지 바늘을 사용하여 +1 ML의 주사기를 T 세포를로드합니다. 주입을위한 준비, 18-1를 교체 ½ 게이지 바늘을 27과 ½ 게이지 바늘을. </li><li> 그들은 행동을 손질 전시 될 때까지 가열 램프 6-8 주 된 여성 NOD.SCID받는 쥐를 노출합니다. </li><li> 고정 장치에서받는 생쥐을 억제하고 주사 부위를 소독 70 % (V / V) 에탄올로 자신의 꼬리를 닦아냅니다. </li><li> 측면 꼬리 정맥 중 하나에 1-2 X 10 6 FACS 분류 세포 / 마우스 (최대 500 μL PBS)로 주입한다. </li></ol> 플런저를 강요하지 않습니다. 바늘이 정맥에 적절하게있는 경우, 주입 거의 저항 일어날 것입니다. 4. 고혈당에 대한받는 사람 쥐 모니터링 및 T1D <ol><li> T 세포 전송 후 5 일부터 NOD.SCID받는 마우스는 제조업체의 지침에 따라 시약 스트립을 (바이엘 Diastix)를 사용하여 높은 소변 포도당 동안 매일 모니터링됩니다. 두 개의 연속 urin와 마우스전자 포도당 수치가&gt; 250 ㎎ / dl로는 당뇨병 간주됩니다. </li></ol>

<ol>
	<li>Makino, S., 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=Breeding+of+a+non-obese,+diabetic+strain+of+mice.">Breeding of a non-obese, diabetic strain of mice.</a> Jikken Dobutsu. 29, 1-13 (1980).</li><li>van Belle, T. L., Coppieters, K. T., von Herrath, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes:+etiology,+immunology,+and+therapeutic+strategies.">Type 1 diabetes: etiology, immunology, and therapeutic strategies.</a> Physiol. Rev. 91, 79-118 (2011).</li><li>Delovitch, T. L., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonobese+diabetic+mouse+as+a+model+of+autoimmune+diabetes:+immune+dysregulation+gets+the+NOD.">The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD.</a> Immunity. 7, 727-738 (1997).</li><li>Wicker, L. S., Miller, B. J., Mullen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+autoimmune+diabetes+mellitus+with+splenocytes+from+nonobese+diabetic+(NOD)+mice.">Transfer of autoimmune diabetes mellitus with splenocytes from nonobese diabetic (NOD) mice.</a> Diabetes. 35, 855-860 (1986).</li><li>Bendelac, A., Carnaud, C., Boitard, C., Bach, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syngeneic+transfer+of+autoimmune+diabetes+from+diabetic+NOD+mice+to+healthy+neonates.+Requirement+for+both+L3T4++and+Lyt-2++T+cells.">Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt-2+ T cells.</a> J. Exp. Med. 166, 823-833 (1987).</li><li>Haskins, K., McDuffie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+diabetes+in+young+NOD+mice+with+a+CD4++islet-specific+T+cell+clone.">Acceleration of diabetes in young NOD mice with a CD4+ islet-specific T cell clone.</a> Science. 249, 1433-1436 (1990).</li><li>Christianson, S. W., Shultz, L. D., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+diabetes+into+immunodeficient+NOD-scid/scid+mice.+Relative+contributions+of+CD4++and+CD8++T-cells+from+diabetic+versus+prediabetic+NOD.NON-Thy-1a+donors.">Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors.</a> Diabetes. 42, 44-55 (1993).</li><li>Wicker, L. S., Todd, J. A., Peterson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+autoimmune+diabetes+in+the+NOD+mouse.">Genetic control of autoimmune diabetes in the NOD mouse.</a> Annual Reviews in Immunology. 13, 179-200 (1995).</li><li>Serreze, D. V., 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=MHC+class+I-mediated+antigen+presentation+and+induction+of+CD8++cytotoxic+T-cell+responses+in+autoimmune+diabetes-prone+NOD+mice.">MHC class I-mediated antigen presentation and induction of CD8+ cytotoxic T-cell responses in autoimmune diabetes-prone NOD mice.</a> Diabetes. 45, 902-908 (1996).</li><li>Milton, M. J., Poulin, M., Mathews, C., Piganelli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation,+maintenance,+and+adoptive+transfer+of+diabetogenic+T-cell+lines/clones+from+the+nonobese+diabetic+mouse.">Generation, maintenance, and adoptive transfer of diabetogenic T-cell lines/clones from the nonobese diabetic mouse.</a> Methods Mol. Med. 102, 213-225 (2004).</li><li>Kurrer, M. O., Pakala, S. V., Hanson, H. L., Katz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+cell+apoptosis+in+T+cell-mediated+autoimmune+diabetes.">Beta cell apoptosis in T cell-mediated autoimmune diabetes.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 213-218 (1997).</li><li>Amrani, A., 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=Perforin-independent+beta-cell+destruction+by+diabetogenic+CD8(+)+T+lymphocytes+in+transgenic+nonobese+diabetic+mice.">Perforin-independent beta-cell destruction by diabetogenic CD8(+) T lymphocytes in transgenic nonobese diabetic mice.</a> J. Clin. Invest. 103, 1201-1209 (1999).</li><li>Dobbs, C., Haskins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+a+T+cell+clone+and+of+T+cells+from+a+TCR+transgenic+mouse:+TCR+transgenic+T+cells+specific+for+self-antigen+are+atypical.">Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical.</a> J. Immunol. 166, 2495-2504 (2001).</li><li>Haskins, K., Portas, M., Bradley, B., Wegmann, D., Lafferty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-lymphocyte+clone+specific+for+pancreatic+islet+antigen.">T-lymphocyte clone specific for pancreatic islet antigen.</a> Diabetes. 37, 1444-1448 (1988).</li><li>Katz, J. D., Wang, B., Haskins, K., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+a+diabetogenic+T+cell+from+genesis+through+pathogenesis.">Following a diabetogenic T cell from genesis through pathogenesis.</a> Cell. 74, 1089-1100 (1993).</li><li>Stadinski, B. D., 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=Chromogranin+A+is+an+autoantigen+in+type+1+diabetes.">Chromogranin A is an autoantigen in type 1 diabetes.</a> Nat. Immunol. 11, 225-231 (2010).</li><li>Luhder, F., Chambers, C., Allison, J. P., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pinpointing+when+T+cell+costimulatory+receptor+CTLA-4+must+be+engaged+to+dampen+diabetogenic+T+cells.">Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 12204-12209 (2000).</li><li>Tang, Q., 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=In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J. Exp. Med. 199, 1455-1465 (2004).</li><li>Calderon, B., Suri, A., Pan, X. O., Mills, J. C., Unanue, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-gamma-dependent+regulatory+circuits+in+immune+inflammation+highlighted+in+diabetes.">IFN-gamma-dependent regulatory circuits in immune inflammation highlighted in diabetes.</a> J. Immunol. 181, 6964-6974 (2008).</li><li>Shultz, L. D., 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=Multiple+defects+in+innate+and+adaptive+immunologic+function+in+NOD/LtSz-scid+mice.">Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.</a> J. Immunol. 154, 180-191 (1995).</li><li>Waldner, H., Sobel, R. A., Price, N., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autoimmune+diabetes+locus+Idd9+regulates+development+of+type+1+diabetes+by+affecting+the+homing+of+islet-specific+T+cells.">The autoimmune diabetes locus Idd9 regulates development of type 1 diabetes by affecting the homing of islet-specific T cells.</a> J. Immunol. 176, 5455-5462 (2006).</li><li>Verdaguer, J., 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=Spontaneous+autoimmune+diabetes+in+monoclonal+T+cell+nonobese+diabetic+mice.">Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.</a> J. Exp. Med. 186, 1663-1676 (1997).</li><li>Thomas, D. C., Mellanby, R. J., Phillips, J. M., Cooke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+early+age-related+increase+in+the+frequency+of+CD4++Foxp3++cells+in+BDC2.5NOD+mice.">An early age-related increase in the frequency of CD4+ Foxp3+ cells in BDC2.5NOD mice.</a> Immunology. 121, 565-576 (2007).</li><li>Prochazka, M., Gaskins, H. R., Shultz, L. D., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonobese+diabetic+scid+mouse:+model+for+spontaneous+thymomagenesis+associated+with+immunodeficiency.">The nonobese diabetic scid mouse: model for spontaneous thymomagenesis associated with immunodeficiency.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 3290-3294 (1992).</li></ol>

모든 마우스는 펜 스테이트 기관 동물 관리의 지침에 따라 및 사용위원회 의과 특정 병원균이없는 (SPF) 시설의 펜실베니아 주립 대학에서 지내게되었다. 저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

T1D은 당뇨병 NOD 마우스 또는 diabetogenic T 세포 클론에서 유래 TCR는 형질 전환 생쥐에서 전체 비장 세포 또는 T 세포 부분 집합의 입양 전송하여 효율성을 다양한에서받는 생쥐에서 유도 할 수 있습니다. 우리는 여기 NOD.SCID 생쥐에 FACS - 정제 CD62L + BDC2.5 형질 전환 된 CD4 + T 세포를 전송하여 100 % 발생에서 2 주 이내에받는 생쥐에서 T1D 유도하는 재생 방법을보고합니다. BDC2.5 T 세?...

NOD 마우스는 자발적으로자가 면역 당뇨병을 개발하고 널리 인간의 T1D 1,2에 대한 동물 모델로 사용되었습니다. NOD 생쥐에서 T1D의 발병 기전은 T와 B 세포에 의해 다음 수지상 세포와 대 식세포에 의한 랑게르한스의 췌장의, 나이, 3-4 주에 시작 침윤이 특징입니다. 비파괴 요정 인슐린 염이 단계는 3 세 ~ 6 개월 명백한 당뇨병의 결과로 인슐린 생산 췌장 β 세포의 느린 진보적 인 파괴에 이르게한다. 4,5 비장 세포, CD4 + 당뇨병 NOD 생쥐에서 6,7 또는 CD8 + 8,9 T 세포의 이동은 섬 반응 T 세포가 T1D 병인에 중요한 역할을한다는 것을 나타내는 면역 NOD 생쥐에서 당뇨병을 중재하기 위해 표시되었습니다. 실험 조건에 따라 당뇨병이 연구의 여러 주에 걸쳐 서서히받는 생쥐에서 개발했습니다. 마찬가지로, 다양한 T 세포 클론은 시간과 비용에 의해 파생diabetogenic T 세포의 배양은 몇 주받는 마우스 7,10로 전송 한 후 당뇨병을 중재하는 것으로보고되었다. CD4 또는 CD8 제한된 diabetogenic T 세포 클론, 여러 실험실에서 파생하는 TCR 표현 형질 전환 생쥐의 가용성이어서는 마우스에서 비장 T 세포가 11-13 사람에게 당뇨병을 전송 할 수 있었던 것으로 나타났습니다. 특히, BDC2.5 NOD 마우스는 chromogranin A, 췌장 베타 세포 14-16에서 단백질 특정 BDC2.5 TCR에 대한 형질 전환합니다. 체외 활성화 또는 효율성에게 11,17-19 다양한에서 신생아 나 면역 결핍 NOD 마우스로 당뇨병을 전송 공공연히 당뇨병 또는 prediabetic BDC2.5 생쥐 해제 활성화 또는 전부를 분별 비장 세포에서의 전송합니다. 우리는 정제 된 형질 전환 CD4를 사용하는 간단한 방법 + 높은 효율과 consiste에서받는 생쥐에서 T1D 유도하는 사전 당뇨병 BDC2.5 생쥐에서 T 세포를 설명NCY. 순진, 섬 항원 특이 CD4 + T 세포의 큰 숫자는 CD4에 대한 형광 활성 세포 정렬 (FACS) 형질 전환 TCR Vβ4 체인을 표현 + CD62L + T 세포에 의해 이러한 생쥐에서 격리됩니다. 정제 된 유전자 변형 T 세포는 그 기능 T 및 B 세포의 부족과 인슐린 염 및 당뇨병 무료 20아르 NOD.SCID 마우스로 작동하지 않는 전송됩니다. 받는 마우스는 T 세포 전송 후 2 주 이내에 신속하게 개발 T1D를 나타내는 소변 포도당의 높은 농도를 모니터링합니다. 이종 특이성과 diabetogenic T 세포를 전송, 우리의 프로토콜은 FACS 정렬 된 CD4 + 거의 독점적으로 diabetogenic BDC2.5 TCR을 표현하는 T 세포를 사용하는 다른 방법과 달리. 자신의 동질성으로 인해, 전송 T 세포 (~ 1X10 6 세포 / 마우스) 만 작은 숫자가 100 % 발생에서 2 주 안에 신속하게 T1D 개발을 위해 필요합니다. 우리의 프로토콜의 또 다른 장점은 irradiatio입니다받는 생쥐의 N은 다른 방법입니다 필요하지 않습니다. 이 방법의 잠재적 인 제한은 모두 CD4와 CD8 T 세포의 하위 집합 또는 특정 당뇨병 CD8 T 세포의 공헌에 대해 조사를 허용하지 않는 것입니다. 설명 프로토콜은 대상 기관에 섬 항원 특이 목 세포의 유도에 개입 순진, monospecific CD4 + T 세포뿐만 아니라 치료 전략에 의해 중재 빠른 T1D 개발, 연구에 유용 할 것입니다.

<table><tbody><tr><td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&amp;alpha;BDC 2.5, Tcr&amp;#946;BDC 2.5)</td><td>JAX</td><td>004460</td><td></td></tr><tr><td>NOD.SCID mice (NOD.CB17-&lt;em&gt;Prkdc&lt;sup&gt;scid&lt;/sup&gt;&lt;/em&gt;/J)</td><td>JAX</td><td>001303</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Themo Scientific</td><td>SH30022.01</td><td></td></tr><tr><td>Bayer Diastix</td><td>Fisher Scientific</td><td>AM2803</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Falcon</td><td>352095</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Sterile surgical tweezers</td><td></td><td></td><td></td></tr><tr><td>Sterile small pair scissors</td><td></td><td></td><td></td></tr><tr><td>Sterile large pair scissors</td><td></td><td></td><td></td></tr><tr><td>70 &amp;mu;m cell strainers</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>35 &amp;mu;m cell strainer cap tubes</td><td>BD Biosciences</td><td>352235</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) buffer</td><td></td><td></td><td>0.15 M NH&lt;sub&gt;4&lt;/sub&gt;Cl, 1 mM KHCO&lt;sub&gt;3, &lt;/sub&gt;0.1 mM Na&lt;sub&gt;2&lt;/sub&gt;EDTA, pH 7.2 in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>BD FACSFlowTM sheath fluid</td><td>BD Biosciences</td><td>342003</td><td></td></tr><tr><td>FACS staining buffer</td><td></td><td></td><td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td></tr><tr><td>Phase contrast microscope</td><td></td><td></td><td></td></tr><tr><td>Trypan blue</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Anti-CD4 (APC) mAb</td><td>Biolegend</td><td>1005616</td><td>clone RM4-5</td></tr><tr><td>Anti-TCR V&amp;#946;4 (FITC) mAb</td><td>BD Biosciences</td><td>553365</td><td>clone KT4</td></tr><tr><td>Anti-CD62L (PE) mAb</td><td>BD Biosciences</td><td>553151</td><td>clone MEL-14</td></tr><tr><td>Cell sorter</td><td>BD Biosciences</td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; BD FACSAria III</td></tr><tr><td>Heat lamp</td><td></td><td></td><td></td></tr><tr><td>Mouse restrainer</td><td></td><td></td><td></td></tr><tr><td>1 ml syringes</td><td>Becton Dickinson</td><td>309602</td><td></td></tr><tr><td>18-1&amp;frac12; gauge needles (sterile)</td><td>Becton Dickinson</td><td>305196</td><td></td></tr><tr><td>27&amp;frac12; gauge needles (sterile)</td><td>Becton Dickinson</td><td>305109</td><td></td></tr></tbody></table>

accelerated type 1 diabetes induction in mice by adoptive transfer of diabetogenic cd4+ t cells

nonobese 당뇨병 (NOD) 마우스 자발적 연령 12 주 후에 면역 당뇨병을 개발하고 인간 1 형 당뇨병 (T1D)의 가장 광범위하게 연구 동물 모델입니다. 조사받는 생쥐의 세포 이동 연구는 T 세포가이 모델 T1D 발병에 중요한 것을 설립했다. 우리는 빠르게 정화의 입양 전송에 T1D 유도하기 위해 여기에 간단한 방법을 설명, 기본 CD4 + NOD.SCID받는 생쥐에 섬 특정 T 세포 수용체 (TCR) BDC2.5위한 형질 전환 전 당뇨병 NOD 생쥐에서 T 세포. 이 기술의 주요 장점은 같은 일 이내에, 수신자의 조사가 필요하지 않으며 T1D의 높은 발병률이 T 세포 전송 후 2 주 안에 이끌어됩니다 diabetogenic T 세포의 분리 및 입양 전송이 완료 될 수 있습니다. 따라서, T1D의 병인과 치료 개입의 연구는 이종 T 세포 집단 또는 복제에 의존하는 방법으로보다 빠른 속도로 진행할 수 있습니다당뇨병 NOD 생쥐에서 파생.

우리의 결과는 췌장 림프절로 차 림프 기관에 집 T 세포에 대한 중요한 CD62L을 표현 형질 전환 BDC2.5 세포의 분리를 보여줍니다. 우리의 연구 결과는 더 빠르게 전송하고 효율적으로 NOD.SCID받는 마우스에 T1D이 monospecific T 세포 인구의 강력한 기능을 보여줍니다. diabetogenic CD4 + BDC2.5 생쥐에서 T 세포의 분리는 그림 2에 표시됩니다. 풀 비장과 림프절에서 약 5 × 10 ...

Watch this Scientific Journal Video about Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells at JoVE.com

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

우리는 섬 항원 특정 기본 CD4 + T 세포의 입양 전송하여 두 주 이내에 생쥐에서 제 1 형 당뇨병 (T1D)를 유도하는 재생 방법을 제공합니다.

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 ...

accelerated-type-1-diabetes-induction-mice-adoptive-transfer

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

16.6K 조회수.  Pennsylvania State University College of Medicine. 우리는 섬 항원 특정 기본 CD4 + T 세포의 입양 전송하여 두 주 이내에 생쥐에서 제 1 형 당뇨병 (T1D)를 유도하는 재생 방법을 제공합니다.

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

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL 마우스의 실험적 면역 Hypophysitis의 유도

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

연구

면역학 및 감염병학

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

사람의 체외에서</em면역 불균형의 초기 국가의 인정을위한 심사 도구를> 억제

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

당뇨병 마우스의 신장 캡슐으로 췌장 아일 레츠의 이식

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

생물학

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

도우미에 의존 Adenoviral 벡터로 인한 유전자의 전송에 의한 당뇨 쥐의 간에서 네오 - 작은 섬 형성

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

의학

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 ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

당뇨병 쥐의 부고환 지방 세포에 이식 된 지지체 지지대 지지대

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

생체공학

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

자가 면역 당뇨병 모델에서 기능 테스트를 위한 항원 특이적 원발성 원발성 T 세포의 고효율 생성

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

CBir1 TCR 형질 전환 CD4+ T 세포의 입양 전달에 의한 장 염증유도

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

생쥐에서 자가면역 당뇨병의 입양 이식 모델에서 이식편 생존의 생물발광 모니터링

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Source: Buchele, V., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58871">Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the induction of intestinal graft-versus-host disease, GvHD, in a lethally-irradiated recipient mouse using allogeneic hematopoietic stem cells and GvHD-inducing splenic alloreactive T lymphocytes. This protocol helps establish a severe acute intestinal GvHD model to understand the mechanism of the disease and develop innovative therapeutic options.

동종 골수 및 비장 세포 이식을 통한 마우스 모델에서 이식편 대 숙주 질환 유도

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. GvHD ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터