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 border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&alpha;BDC 2.5, Tcr&beta;BDC 2.5)</td>
			<td>JAX</td>
			<td>004460</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NOD.SCID mice (NOD.CB17-Prkdcscid/J)</td>
			<td>JAX</td>
			<td>001303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
			<td>Themo Scientific</td>
			<td>SH30022.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bayer Diastix</td>
			<td>Fisher Scientific</td>
			<td>AM2803</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile surgical tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile small pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile large pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70 &mu;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 &mu;m cell strainer cap tubes</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium-Chloride-Potassium (ACK) buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2 in dH2O</td>
		</tr>
		<tr>
			<td>BD FACSFlowTM sheath fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS staining buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</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&beta;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>&nbsp;</td>
			<td>e.g. BD FACSAria III</td>
		</tr>
		<tr>
			<td>Heat lamp</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18-1&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td>&nbsp;</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

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

준비 및 사용 HIV - 1 차 감염된 CD4 + 대상 세포로 T 세포 자연 킬러 세포 세포 독성 Assays에서

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

연구

면역학 및 감염병학

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 감염 CD4의 타고난 감지 평가<sup&gt; +</sup&gt;를 사용하여 형질 수지상 세포에 의한 T 세포<em&gt; 예 생체 내</em&gt; 공동 문화 시스템.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

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

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

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

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

CD4의 준비<sup&gt; +</sup&gt; 현미경으로 GD3 및 GD2 강글 리오 시드 멤브레인 식의 분석을위한 T 세포

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

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

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

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

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

TGF-β 함유 프로토콜을 사용하여 나이브 CD4 + T 세포에서 인간의 CD4 + Foxp3의 + 유도 규제 T 세포 (iTregs)의 체외 분화

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

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

마비 및 비주얼 시스템 부상 Neuromyelitis Optica Autoantigen Aquaporin-4에 대 한 T 세포 관련 하 여 쥐에서의 유도

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

절연 및 높은 소금의 입양 전송 처리 수지상 세포를 항 원 제시

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

카르복시플루아세인 수치니미딜 에스테르를 이용한 증식 인간 항원 특이적 CD4+ T 세포의 정량화

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

포도당-6-인산염 이사머라제(G6PI)-유도 RA 마우스에서 iNKT 세포의 입양 면역 요법

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

제1형 당뇨병, 체강 질병 및 COVID-19에 대한 고처리량 멀티플렉스 스크리닝

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and ...