저자는이 프로토콜의 개발에 도움을 젠킨스 연구소의 앙드레 한과 로렌스의 엔 기술 지원 및 회원 감사드립니다.

1. 림프 조직에서 세포 절연 <ol><li> 의 작은 사각형이 포함 된 60mm 문화 요리에, 얼음 차가운 cEHAA (EHAA + 10% FBS, 펜 / 패 혈성, gentamycin, 2 MM L-글루타민, 55 MM 2 메르 캅토 에탄올) 또는 기타 이에 상응하는 T 세포 매체 1 ML을 추가 얼음에 100 μm 나일론 메쉬 및 장소. </li><li> 마우스를 안락사시켜야. </li><li> 비장 가능한 많은 쉽게 접근 할 수 림프절을 제거합니다. 이러한 적어도 사타구니, 겨드랑이, 상완, 자궁 경부, 그리고 장간막 림프절을 포함해야합니다. 배양 접시에있는 나일론 메쉬의 상단에 올려 놓으십시오. </li><li> 부드럽게 덧는 림프구를 해방하기 위해 나일론 메쉬 동안 폐쇄 1.5 ML의 microfuge 튜브의 평면 상단을 림프 조직 사용합니다. 정지에 셀을 작업 할 cEHAA와 pipet 위아래로 다른 한 ML을 추가합니다. 15 ML의 폴리 프로필렌 원심 분리기 튜브의 맨 위에 위치 나일론 메쉬의 또 다른 작품을 통해 셀을 전송합니다. 얼음 차가운 cEHAA, PO의 또 다른 한 ML 함께 그릇과 메쉬를 씻어같은 관에 볼륨을 oling. 4 ML의 최종 세포 현탁액 볼륨을 달성하기 위해 반복합니다. </li><li> 15 ML의 최종 볼륨 (PBS + 2% FBS, 0.05 % Azide) 차가운 정렬 버퍼를 추가, 4, 300 XG에서 5 분 동안 튜브를 원심 분리기 ° C. </li><li> 조심스럽게 액체의 어떤 물방울이 튜브의 측면에 남아되지 않습니다 확실하게, 표면에 뜨는을 대기음. 약 두 번 펠렛 자체의와 동일한 최종 볼륨으로 FC 블록 (정렬 버퍼 + 2.4G2 항체)의 세포 펠렛을 Resuspend. 예를 들어, 순진한 마우스 비장과 림프 노드는 일반적으로 약 100 μl의 세포 펠렛을 생산하고 있습니다. 이 경우, 200 μl의 볼륨을 가져 FC 블록 100 μl를 추가합니다. 셀 거리며 걷게의 큰 학위가 발생했습니다 경우, 조심스럽게 pipet 팁을이 시점에서 세포 덩어리를 제거합니다. </li></ol> 2. Tetramer의 착색 <ol><li> 10 나노 미터 (또는 경험적으로 최적화 된 농도)의 최종 농도에 PE 또는 APC-라벨 pMHC tetramer를 추가합니다. </li><li> 믹스와 incuba실내 온도 (또는 경험적으로 최적화 시간 및 온도)에서 1 시간에 테. </li><li> 15 ML의 볼륨에 감기 정렬 버퍼를 추가하고 300 XG, 4 ° C.에서 5 분 동안 튜브를 원심 분리기 얼음 또는 ° C 지금부터 4에 샘플을 보관. </li><li> 조심스럽게 액체의 어떤 물방울이 튜브의 측면에 남아되지 않습니다 확실하게, 표면에 뜨는을 대기음. 200 μl의 최종 볼륨으로 정렬 버퍼에있는 셀 펠렛을 Resuspend. 더블 tetramer 농축를 들어, 150 μl의 최종 볼륨 resuspend. </li></ol> 3. 자기 강화 <ol><li> Miltenyi 방지 PE 또는 안티 APC microbeads의 50 μl를 추가합니다. 더블 tetramer 농축를 들어, 50 μl를 추가합니다. </li><li> 4에 섞어서 20 분에 품다 ° C. </li><li> 15 ML의 볼륨에 감기 정렬 버퍼를 추가하고 300 XG, 4 ° C.에서 5 분 동안 튜브를 원심 분리기 </li><li> 기다리는 동안 MidiMACS 또는 QuadroMACS 자석에 Miltenyi LS 자기 열을 설정합니다. 에 문이 열려있는 15 ML의 폴리 프로필렌 원심 분리기 튜브의 위치를열 아래에 직접 올릴 수. </li><li> 가 15 ML 튜브로 배수 할 수 있도록, 컬럼의 상단에 정렬 버퍼 3 ML을 추가합니다. </li><li> 칼럼의 상단에 100 μm 나일론 메쉬 광장을 배치합니다. </li><li> 세포가 회전이 완료되면, 조심스럽게 표면에 뜨는을 기음과 정렬 버퍼 3 ML에서 펠렛을 resuspend. </li><li> 열 상단 위에 나일론 메쉬를 통해 세포 현탁액을 전송합니다. </li><li> 세포 현탁액이 완전히 열을 배출되면, 정렬 버퍼의 다른 3 ML로 원래 튜브를 씻어 열로 나일론 메쉬를 통해 버퍼를 전송 과정에서 메쉬를 린스. 나일론 메쉬를 폐기하십시오. </li><li> 버퍼가 완전히 열을 배출되면 열을 정렬 버퍼의 또 다른 3 ML를 추가합니다. </li><li> 3 × 3 ML 세차 총 10 단계를 반복합니다. </li><li> 새로운 15 ML의 폴리 프로필렌 원심 분리기 튜브를 통해 자석과 장소에서 열을 제거합니다. </li><li> t로 정렬 버퍼의 5 ML을 추가그는 열. </li><li> 즉시 열 상단에 플런저를 삽입하고 하나의 연속 동작으로, 튜브에 열을 밖으로 버퍼를 강제, 플런저 모든 방법을 아래로 밀어. </li><li> 300 XG에서 5 분의 용출 바인딩 된 분수와 흐름을 통해 언 바운드 분수, 4 ° C.를 포함하는 튜브를 원심 분리기 </li><li> 액체의 더 작은 물방울이 튜브의 측면에 남아되지 않습니다 확실하게, 바인딩 분수에서 표면에 뜨는을 자세히 대기음. 정확히 95 μl의 최종 볼륨으로 정렬 버퍼에있는 셀 펠렛을 Resuspend. 기음과 2 ML의 최종 볼륨에 언 바운드 일부를 resuspend. </li></ol> 4. 유동 세포 계측법 <ol><li> 각 분수를 들어, 5 μl를 제거하고 5 ML의 FACS 관 계수 구슬 200 μl (정렬 버퍼에 200,000 / ML의 농도로 조정)에 추가 할 수 있습니다. 4에 정해 ° C를 분석하기. 시간을 절약하기 위해 구슬은 실험 시작 전에 레이블 튜브에 미리 aliquoted 할 수 있습니다. </li><li> 오전 준비항체의 애 스터 믹스는 세포의 표면 마커 (표 1) 얼룩합니다. 시간을 절약하기 위해, 이것은 실험의 시작하기 전에 수행 할 수 있습니다. </li><li> 바인딩 된 분수를 들어, 관 세포 ~ 90 μl에 직접 항체 칵테일의 복용을 추가합니다. 언 바운드 분수의 분석이 필요하다면, 5 ML의 FACS 튜브에 세포의 90 μl를 전송하고 항체 칵테일의 복용을 추가합니다. </li><li> 흐름 cytometer위한 단일 색상 보정 컨트롤 패널을 설정합니다. 사용 할 각각의 fluorochrome를 들어, 해당 fluorochrome에 복합 항 CD4 항체 1 μl로 5 ML의 FACS 관에 남은 언 바운드 분획 세포의 50 μl를 섞는다. 뿐만 아니라 흠없는 제어를 설정해야합니다. </li><li> 소용돌이와 4에서 30 분의 모든 샘플을 품다 ° C. </li><li> 300 XG에서 5 분, 4 ° C. 각 튜브와 원심 분리기로 정렬 버퍼의 5 ML을 추가 </li><li> 바인딩 된 분수를 들어, 신중하게 정렬 buf 200 μl의 세포 표면에 뜨는 기음 및 resuspend남았다. 1.2 ML의 FACS의 microtube에 세포를 전송합니다. 같은 microtube에 정렬 버퍼와 수영장의 또 다른 200 μl로 관을 씻어. 셀 clumps이 명백 경우, 50 μm 필터를 통해 세포를 전달합니다. </li><li> 언 바운드 분율 및 보상 제어, 가만히 따르다 또는의 표면에 뜨는 및 resuspend 두 ML의 정렬 버퍼를 대기음하십시오. </li><li> 단일 색 보정 컨트롤을 사용하여 흐름 cytometer를 설정합니다. 기록 할 추가 매개 변수로 측 분산 폭 (SSC-W)를 선택합니다. </li><li> 도 1 및도 2에 도시 된 바와 같이 + 나 CD8 + T 세포 CD4을 식별 할 연속 포함 된 문의 순서를 사용하여 스테인드 샘플을 분석합니다. 바인딩 분수를 들어, 최대 2,000,000 총 이벤트의 최대 가능한 한 많은 세포를 수집합니다. 언 바운드 분수를 들어, 1,000,000 총 이벤트를 수집합니다. 수집 속도 초당 이벤트이나 3000 아래하세요. </li><li> 같은 시스템 설정을 사용하여 계산 비드 샘플을 분석 할 수 있습니다. 10,000 총 이벤트를 수집합니다. &lt;/ 리&gt; <li> FCS 파일로 모든 데이터를 저장합니다. </li></ol> 5. 데이터 분석 <ol><li> FlowJo 소프트웨어를 사용하여 계산 비드 샘플에 대한 FCS 데이터 파일을 분석합니다. 플롯 앞으로 FITC의 분산 및 (그림 1과 2 참조) 계산 구슬을 aroung 게이트를 설정합니다. 각 샘플에서 발견 비즈를 계산의 총 수를 결정합니다. 수집 셀 이벤트의 수를 결정하기 위해 수집 이벤트의 총 수에서 뺍니다. </li><li> 박스 1에서 설명한 방정식을 사용하여 각 샘플에있는 모든 세포의 총 수를 계산합니다. </li><li> 스테인드 바운드 및 언 바운드 일부 샘플에 대한 FCS 데이터 파일을 분석합니다. 림프 +, 측면 산란 - 너비 이오, 덤프 -, 3 번 CD +, CD4 + 또는 CD8 +, tetramer도 1 및도 2에 도시 된 바와 같이 각 예제에서 + T 세포.을 식별 할 연속 포함 된 문의 순서를 설정 </li><li> CD4을 정의하는 데 사용 포함 된 문이 각각의 주파수 + tetramer하여 샘플에서 세포의 총 수를 모두 곱하면+ 또는 CD8 + tetramer + 세포 에피토프 특정 T 세포 (박스 1)의 총 수를 계산합니다. </li></ol>

<ol>
	<li>Knutson, K. L., dela Rosa, C., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+analysis+of+T-cell+immunity.">Laboratory analysis of T-cell immunity.</a> Front Biosci. 11, 1932-1944 (2006).</li><li>Kearney, E. R., Pape, K. A., Loh, D. Y., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+peptide-specific+T+cell+immunity+and+peripheral+tolerance+induction+in+vivo.">Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo.</a> Immunity. 1, 327-339 (1994).</li><li>Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+epitope-specific+T+cells.">Tracking epitope-specific T cells.</a> Nat Protoc. 4, 565-581 (2009).</li><li>Hataye, J., Moon, J. J., Khoruts, A., Reilly, C., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+and+memory+CD4++T+cell+survival+controlled+by+clonal+abundance.">Naive and memory CD4+ T cell survival controlled by clonal abundance.</a> Science. 312, 114-116 (2006).</li><li>Marzo, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+T+cell+frequency+dictates+memory+CD8++T+cell+lineage+commitment.">Initial T cell frequency dictates memory CD8+ T cell lineage commitment.</a> Nat Immunol. 6, 793-799 (2005).</li><li>Davis, M. M., Altman, J. D., Newell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+the+repertoire:+broadening+the+scope+of+peptide-MHC+multimer+analysis.">Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis.</a> Nature reviews. Immunology. 11, 551-558 (2011).</li><li>Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+CD4(+)+T+cell+frequency+varies+for+different+epitopes+and+predicts+repertoire+diversity+and+response+magnitude.">Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.</a> Immunity. 27, 203-213 (2007).</li><li>Seah, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linear+range+for+accurately+quantifying+antigen-specific+T-cell+frequencies+by+tetramer+staining+during+natural+immune+responses.">The linear range for accurately quantifying antigen-specific T-cell frequencies by tetramer staining during natural immune responses.</a> European Journal of Immunology. 41, 1499-1500 (2011).</li><li>Obar, J. J., Khanna, K. M., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+naive+CD8++T+cell+precursor+frequency+regulates+primary+and+memory+responses+to+infection.">Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection.</a> Immunity. 28, 859-869 (2008).</li><li>Daniels, M. A., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+for+CD8+in+T+cell+receptor+binding+and+activation+by+peptide/major+histocompatibility+complex+multimers.">Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.</a> J Exp Med. 191, 335-346 (2000).</li><li>Pittet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha+3+domain+mutants+of+peptide/MHC+class+I+multimers+allow+the+selective+isolation+of+high+avidity+tumor-reactive+CD8+T+cells.">Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.</a> Journal of Immunology. 171, 1844-1849 (2003).</li><li>Choi, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+avidity+antigen-specific+CTL+identified+by+CD8-independent+tetramer+staining.">High avidity antigen-specific CTL identified by CD8-independent tetramer staining.</a> Journal of Immunology. 171, 5116-5123 (2003).</li><li>Chu, H. H. <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+optimizes+the+number+and+function+of+MHCII-restricted+CD4++T+cell+clones+in+the+naive+polyclonal+repertoire.">Positive selection optimizes the number and function of MHCII-restricted CD4+ T cell clones in the naive polyclonal repertoire.</a> Proc Natl Acad Sci U S A. 106, 11241-11245 (2009).</li><li>Chu, H. H., Moon, J. J., Kruse, A. C., Pepper, M., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Selection+and+Peptide+Chemistry+Determine+the+Size+of+Naive+Foreign+Peptide-MHC+Class+II-Specific+CD4++T+Cell+Populations.">Negative Selection and Peptide Chemistry Determine the Size of Naive Foreign Peptide-MHC Class II-Specific CD4+ T Cell Populations.</a> J Immunol. 185, 4705-4713 (2010).</li><li>Legoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+TCR+reactivity+and+HLA+phenotype+on+naive+CD8+T+cell+frequency+in+humans.">Impact of TCR reactivity and HLA phenotype on naive CD8 T cell frequency in humans.</a> J Immunol. 184, 6731-6738 (2010).</li><li>Alanio, C., Lemaitre, F., Law, H. K., Hasan, M., Albert, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+human+antigen-specific+naive+CD8++T+cells+reveals+conserved+precursor+frequencies.">Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies.</a> Blood. 115, 3718-3725 (2010).</li><li>Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+Epitope-Specific+Naive+CD4++T+Cells+Correlates+with+Immunodominance+in+the+Human+Memory+Repertoire.">Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire.</a> Journal of Immunology. 188, 2537-2544 (2012).</li><li>Jenkins, M. K., Chu, H. H., McLachlan, J. B., Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+composition+of+the+preimmune+repertoire+of+T+cells+specific+for+Peptide-major+histocompatibility+complex+ligands.">On the composition of the preimmune repertoire of T cells specific for Peptide-major histocompatibility complex ligands.</a> Annu Rev Immunol. 28, 275-294 (2010).</li><li>Matechak, E. O., Killeen, N., Hedrick, S. M., Fowlkes, B. J. <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+II-specific+T+cells+can+develop+in+the+CD8+lineage+when+CD4+is+absent.">MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent.</a> Immunity. 4, 337-347 (1996).</li><li>Burchill, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linked+T+cell+receptor+and+cytokine+signaling+govern+the+development+of+the+regulatory+T+cell+repertoire.">Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire.</a> Immunity. 28, 112-121 (2008).</li><li>Pepper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+routes+of+bacterial+infection+induce+long-lived+TH1+memory+cells+and+short-lived+TH17+cells.">Different routes of bacterial infection induce long-lived TH1 memory cells and short-lived TH17 cells.</a> Nature Immunology. 11, 83-89 (2010).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 프로토콜에서 제공 pMHC tetramer 기반의 세포 농축 방법은 내생 T 세포 repertoires에서 에피토프 특정 T 세포를 연구하기위한 강력한 도구입니다. pMHC의 tetramers의 사용은 직접적으로 기원 pMHC 리간드를 구속하기 위해 자신의 TCRs의 능력에 따라 에피토프 특정 T 세포의 확인을 할 수 있습니다. 강화는 항원에 특정한 T 세포의 매우 희귀 한 인구는 자신의 유전 메이크업이나 전구체 주파수의 조작없이 T ...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; 시약</strong</td&gt;<td<strong&gt; 공급 업체</strong</td&gt;<td<strong&gt; 카탈로그 번호</strong</td&gt;</tr&gt;<tr&gt;<td&gt; PE 또는 APC 복합 pMHC tetramer (또는 multimer)</td&gt;<td&gt; NIH tetramer 코어에서 얻은, 조사원에 의해 제작, 또는 상업적 소스에서 구입</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 안티 - PE 복합 자성 microbeads</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-048-801</td&gt;</tr&gt;<tr&gt;<td&gt; 안티 - APC 복합 자성 microbeads</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-090-855</td&gt;</tr&gt;<tr&gt;<td&gt; LS 자기 열</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-401</td&gt;</tr&gt;<tr&gt;<td&gt; MidiMACS 또는 QuadroMACS 자석</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-302 또는 130-090-976</td&gt;</tr&gt;<tr&gt;<td&gt; 셀 계산 비즈</td&gt;<td&gt; 생명 기술</td&gt;<td&gt; PCB-100</td&gt;</tr&gt;</tbody&gt;</table

peptide:mhc tetramer-based enrichment of epitope-specific t cells

생체 실험 모델과 적응 면역을 공부 연구자에 대한 기본적인 필요는 T 세포 항원 수용체 (TCR) 특이성에 따라 T 세포를 식별 할 수 있다는 것입니다. 많은 간접 방법은 T 세포의 대량 인구가 특정 항원과 에피토프 특정 T 세포 등 확산, 시토 킨 생산, 또는 활성 마커 하나의 표현과 같은 기능 응답의 측정을 통해 확인할 수 있습니다와 체외에서 자극되는 이용하실 수 있습니다. 그러나, 이러한 방법은 여러 가지 기능 중 하나를 전시 에피토프 특정 T 세포를 식별, 그들은 순진한 전구체 주파수에서 에피토프 특정 T 세포를 감지 할 수있을만큼 문자를 구분하지 않습니다. 인기있는 대안은, TCR 유전자 변형 입양 이전 모델 인에서 TCR 유전자 변형 마우스의 단클론 T 세포는 easi 수 있습니다 에피토프 특정 T 세포의 큰 전구체 인구를 만들 histocompatible 호스트에 놓는 아르통증은 congenic 마커 항체 2,3의 사용을 추적. 강력한 있지만,이 방법은 4,5 하나의 에피토프에 대한 특이성과 T 세포의 unphysiological 주파수와 관련된 실험 유물을 앓고. 또한,이 시스템은 polyclonal 인구에서 에피토프 특정 T 세포 클론의 기능 이질성을 조사하는 데 사용할 수 없습니다. MHC (pMHC) 단지 : 적응 면역을 연구 할 수있는 이상적인 방법은 전적으로 해당 펩타이드 기원에 바인딩하여 TCR의 특이성을 구별하는 방법을 사용하여 내생 T 세포 레퍼토리의 에피토프 특정 T 세포의 직접 검출을 포함해야합니다. pMHC tetramers 및 유동 세포 계측법의 사용이 6 달성 있지만 다음과 같은 항원 유발 clonal 확장을 발견 에피토프 특정 T 세포의 높은 주파수 인구의 감지로 제한됩니다. 이 프로토콜에서는, 우리는 pMHC의 tetramers의 사용을 조정 방법 및 magne을 설명틱 세포 농축 기술은 마우스 림프 조직 3,7에서 매우 낮은 주파수 에피토프 특정 T 세포의 탐색 기능을 사용하도록 설정합니다. 이 기술로, 하나는 종합적으로 면역 반응의 모든 단계에서 쥐 내생 T 세포의 전체 에피토프 별 인구를 추적 할 수 있습니다.

그림 2는 이전에 관련 펩타이드 + CFA와 함께 예방 접종을 쥐 대표 데이터를 묘사하면서 그림 1은 순진 쥐에서 pMHCII tetramer 강화 비장과 림프절 샘플을 대표하는 유동 세포 계측법의 음모를 도시한다. 시리얼 게이트는 CD4의 분석 + T 세포 인구에서 autofluorescent 및 기타 원치 않는 이벤트를 제거합니다. CD8 + T 세포 인구는 CD4의 pMHCII tetramer의 착색 + T 세포에 유용한 내부 대조군...

Watch this Scientific Journal Video about Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells at JoVE.com

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

에피토프 특정 T 세포의 낮은 주파수 인구를 분리하고 유동 세포 계측법을 기준으로 결과를 분석 MHC tetramers 및 자기 microbeads :이 프로토콜은 펩타이드의 사용을 설명합니다. 이 방법은에서 관심 내생 T 세포 인구의 직접적인 연구를 수<em&gt; 생체 내</em&gt; 실험 시스템.

펩타이드 : 에피토프 특정 T 세포의 MHC Tetramer 기반 강화

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

peptidemhc-tetramer-based-enrichment-of-epitope-specific-t-cells

Research

JoVE Journal

Immunology and Infection

17.8K 조회수.  Massachusetts General Hospital and Harvard Medical School. 에피토프 특정 T 세포의 낮은 주파수 인구를 분리하고 유동 세포 계측법을 기준으로 결과를 분석 MHC tetramers 및 자기 microbeads :이 프로토콜은 펩타이드의 사용을 설명합니다. 이 방법은에서 관심 내생 T 세포 인구의 직접적인 연구를 수 생체 내 실험 시스템.

비디오: Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

연구

면역학 및 감염병학

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

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

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

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

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

piggyBac Transposon System Modification of Primary Human T Cells

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

기본 인간 T 세포 piggyBac Transposon 시스템 변경

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

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T 세포의 유도<em&gt; 체외</em&gt; 마우스 배아 줄기 세포에서

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

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

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

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

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

사이토 카인 캡처 시스템을 사용하여 임상 응용 프로그램에 대한 거대 세포 바이러스 고유의 자동화 된 세포 농축은 T 세포

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

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

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

T 세포는 수지상 세포에서 Transinfection에 의해 박테리아를 캡처

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

Production of Human CRISPR-Engineered CAR-T Cells

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

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

인간 CRISPR-엔지니어링 CAR-T 셀 생산

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Adoptive Transfer of P14 T-Cells into Tumor-Bearing Mice

P14 T-세포를 종양이 있는 마우스로 입양 이식

Isolation and Characterization of Antigen-Specific T cells in Mouse Lungs(마우스 폐에서 항원 특이적 T 세포의 분리 및 특성 분석)

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

저자 스포트라이트: Tetramers를 사용하여 마우스 폐에서 항원 특이적 T 세포를 검출하기 위한 최적화된 프로토콜

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

펩타이드를 이용한 생쥐 폐의 희귀 항원 특이적 T 세포 식별: 주요 조직적합성 복합체 테트라머

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...