The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

참고 :이 프로토콜은 인간의 자원 봉사자 말초 혈액 샘플의 사용이 필요합니다. 인간을 대상으로 모든 연구를 검토하고 헬싱키 선언 (2013)과 건강 보험 양도 및 1996 년 책임에 관한 법안을 준수하기 위해 인간 연구 윤리 심의위원회의 승인을 받아야한다. 전혈에서 T 세포의 분리 1. <ol><li> 시작에 앞서, 실온 밀도 구배 매질을 따뜻하게. 나누어 2-4 멸균 15 ml의 원추형 원심 분리 튜브에 밀도 구배 배지 4 ㎖ (1 관 각각 10 ml의 희석 된 혈액의 총량에 대해 사용된다). </li><li> 1-2 나트륨 헤파린 스프레이 혈액의 10 ~ 20 ml의 코팅 (녹색 위) 정맥 혈액 수집 튜브를 얻습니다. 교육을받은 사람의 감독하에 인간의 표본을 수집하고 심사위원회 (Institutional Review Board)에 따라 프로토콜을 승인했다. </li><li> 70 % 에탄올로 튜브의 외부를 닦으십시오. 조심스럽게 Fi의 꼭대기를 제거수집 튜브를 채워하고 멸균 50 ㎖ 원심 분리 튜브로 혈액을 가만히 따르다. </li><li> 각각의 혈액 수집 튜브에 10 ㎖의 멸균 인산 완충 식염수 (PBS)를 첨가. , PBS를 수집 경사 전체 혈액에 넣어 천천히 섞는다. </li><li> 25 μL 인간 T 세포 농축 칵테일 / 2 ml의 전체 볼륨을 추가합니다. 20 분 동안 실온에서 인큐베이션. </li><li> 부드럽게 밀도 구배 배지 위에 한 혈액 희석 : 10 ㎖를 피펫을 사용하여, (1) 10 ㎖까지 층. 밀도 기울기 매체의 표면을 방해하지 않도록주의하십시오. </li><li> 20 오 C. 원심 분리기 계층화 혈액 및 밀도 구배 매체를 20 분 동안 1,200 XG에 </li><li> 밀도 구배 매질 및 밀도 구배 매체 희석 플라즈마 사이의 계면에서 백혈구의 층 사이의 계면을 방해하지 않도록주의하면서, 원심 분리기에서 튜브를 제거한다. 5 ml의 피펫을 사용하여 조심스럽게 백혈구 층을 수집하고 새로운 무균 50 ㎖ 원뿔형 욕조로 전송할전자. </li><li> 50ml로 수집 된 PBL의 볼륨을 가지고 부드럽게 섞어 PBS를 추가합니다. </li><li> 20 O ℃에서 5 분 동안 600 XG에 원심 분리기 가만히 따르다. </li><li> 이 재현 탁 완충액 (PBS 멸균 여과는 1 % BSA 함유) 정렬 계측법 10 ㎖의 멸균 유동 세포를 펠렛 화. 샘플 세포 현탁액 10 μL, 1:10 희석는 혈구 세포를 사용하는 계산 트리 판 블루 (90 μL에 추가) (예상 수익률을 1-4 × 10 6 세포 전체 혈액의 / 튜브). <ol><li> 5 ML 튜브로 전송, 1 ml의 나누어지는 제거, 사량으로 표시하지 T 세포의 분석을 위해 얼음에 보관하십시오. 얼음에 세포 현탁액을 유지합니다. </li></ol></li></ol> Alloantigen 특정 T 세포의 2 마그네틱 심화 <ol><li> 1 alloantigen pMHC의 사량을 희석 : (100)를 멸균 종류의 버퍼에. </li><li> 원심 분리기에서 세포 현탁액 20 오 C.에서 5 분 동안 600 XG에 (단계 1.11 9 ml) 중 가만히 따르다. </li><li> 펠렛에 pMHC 량체 alloantigen 희석의 50 μl를 추가세포 (10 7 세포까지). 부드러운 피펫 팅으로 섞는다. 멸균 5 ML 튜브로 전송합니다. 실온에서 30 분 동안 인큐베이션. </li><li> 일종의 버퍼 2 ML을 추가합니다. 20 O ℃에서 5 분 동안 600 XG에 원심 분리기 가만히 따르다. </li><li> 100 μL 종류의 버퍼에 재현 탁 세포. 10 μL 비오틴 선택 칵테일을 첨가하고, 실온에서 15 분 동안 배양한다. </li><li> 5 ㎕의 자성 나노 입자를 첨가하고, 실온에서 10 분 동안 배양한다. </li><li> 2.5 ml의 정렬 버퍼와 피펫으로 부드럽게 혼합 추가합니다. 5 ML 튜브로 전송, 100 μL 나누어지는 제거 및 사전 농축 세포의 분석을 위해 얼음에 보관하십시오. </li><li> 세포 분리 자석에 세포 현탁액을 함유하는 5 ㎖의 튜브를 놓는다. 캡 튜브 위에 느슨하게해야한다. 실온에서 5 분 동안 인큐베이션. </li><li> 부드럽게 튜브의 캡을 제거합니다. 함께 튜브와 자석을 잡고, 신선한 5 ML 튜브로 튜브의 내용을 가만히 따르다. 누르거나 튜브의 내용을 흔들지 마십시오액체의 마지막 방울을 제거합니다. </li><li> 자석에서 튜브를 제거합니다. 2 ml의에게 차가운 종류의 버퍼를 추가하고 피펫으로 가볍게 섞는다. 이 혈구를 사용하여 세포를 계산하는 트리 판 블루 (10 μL에 추가) : 샘플 세포 현탁액 10 μL은, (1) 희석. </li></ol> 단일 셀 유동 세포 계측법 셀 정렬에 대한 T 세포의 3. 준비 <ol><li> 세포의 모든 튜브에 차가운 종류의 버퍼를 추가 (사량 레이블, 사량 표지 농축 - 유엔, 사량 표지 농축) 3 ㎖에 볼륨을 가지고. </li><li> 4 O C에서 5 분 600 XG에 원심 분리기 및 캔트 버퍼는 세포를 복구 할 수 있습니다. </li><li> 25 μl의 차가운 종류의 버퍼에 펠렛 세포를 재현 탁. 5 μL 인간의 Fc 블록을 추가합니다. 20 분 동안 얼음에 품어. </li><li> 유동 세포 계측법 정렬에 대한 항체를 준비합니다. 15 μL 정렬 버퍼, 15 μL 안티 CD5, 15 μL 항 CD14, 15 μL 항 CD19를 섞는다. </li><li> 세포의 각 튜브에 항체 혼합물의 20 μl를 추가합니다. 20 분 동안 얼음에 품어. </li><li> 각각의 튜브에 차가운 종류의 버퍼의 2 ML을 추가합니다. </li><li> 4 O C에서 5 분 600 XG에 원심 분리기 및 캔트 버퍼는 세포를 복구 할 수 있습니다. </li><li> 정렬 버퍼에 1~2 × 106 세포 / ml의 농도로 세포를 재현 탁. </li><li> 인간 T 세포 배양 배지의 분취 량 100 ㎕를가 (Iscove의 DMEM 2 mM의 루타, 10 mM의 HEPES, 50 ㎍ / ml의 겐타 마이신, 50 μM 2- 머 캅토 에탄올, 10 % 열 - 불 활성화 된 인간 AB 혈청, 2.5 NG / ml의 재조합 인간 보충 IL-2)를 96 웰 둥근 바닥 세포 배양 플레이트의 각 웰에. 얼음에 보관하십시오. </li></ol> 단일 셀 유동 세포 계측법 정렬하여 테트라 머 표지 T 세포 4. 분리 <ol><li> alloantigen-pMHC 량체 표시된 T 세포 (그림 2.A)를 식별하는 게이팅 매개 변수 유동 세포 계측법을 설정합니다. B 세포와 CD14를 발현하는 CD19를 제외, 림프구에 이중선, 게이트를 제거하는 전략을 게이팅 설정하기 위해 사전 농축 사량 표시된 부분을 사용하여단핵구를 발현하고, CD5 식에 의해 T 세포를 식별. (사량으로 표시하지 샘플에서 세포가이 게이트 내에해야한다 - - CD19 CD5 + CD14의 0 %로) 사량 결합 T 세포의 식별을위한 게이팅을 설정하는 형광으로 사량을 뺀 컨트롤을 표시하지 샘플을 사용 . NOTE : 지나치게 제한 게이팅 전략 절연 T 세포의 수를 줄이는 반면, 특정 항원되지 않은 T 세포 또는 T 세포의 분리 될 것이다 충분히 엄격한 아닌 전략을 게이팅. </li><li> 단일 세포 종류의 플레이트 매개 변수를 프로그래밍합니다. CD19 - - 각 웰의 사량 + 셀 (1) CD5 + CD14을 배치 할 분류기을 지시. 각 행의 (그림 2.B 잘 (세포 - CD19 - - 사량 100 CD5 + CD14)을 잘 한 음성 대조군 (어떤 세포가 우물에 분류 없음)과 1 개의 긍정적 인 제어를 포함하도록 설정 판을 직접 </strong&gt;). </li><li> 게이팅 전략 및 플레이트 셋업 계측법 확립 흐름을 사용하여, 직접 유세포 셀 소터를 사용하여 96 웰 플레이트에 각각의 사량 체 - 결합 T 세포를 분리. </li></ol> 5. 문화 및 Alloantigen 특정 T 세포 클론의 확장 <ol><li> 20 O ℃에서 세포 분류, 5 분 600 XG에 원심 분리기 판의 완료 후 6 % CO 2 배양기에서 37 O C에서 개최 배양 플레이트. </li><li> 30 초 동안 소용돌이 자극 항 CD3 / 안티 CD28 마이크로 비드는 구슬을 재현 탁합니다. </li><li> 수집 된 T 세포의 자극에 필요한 활성제 비드 부피 (활성제 마이크로 비드 / 웰의 0.5 μl를) 계산한다. 멸균 5 ML 튜브에 자극 구슬의 계산 된 볼륨을 전송합니다. 1 ml의에게 인간 T 세포 배양 배지와 소용돌이를 추가합니다. </li><li> 자석의 마이크로 비드 서스펜션 튜브를 놓습니다. 캡 튜브 위에 느슨하게해야한다. 실온에서 2 분 동안 인큐베이션. </li><li> 부드럽게 제거튜브의 캡. 신선한 5 ML 튜브에 함께 캔트 튜브의 내용을 튜브와 자석을 들고. 누르거나 튜브의 내용을 흔들지 마십시오. </li><li> 자석에서 튜브를 제거합니다. 인간 T 세포 배양 배지를 추가 (마이크로 비드 100 ㎕ 매체 / 0.5 μL는 처음에 추가). </li><li> 나누어 96 웰 플레이트의 각 웰에 활성제 비드 현탁액 100 ㎕. 6 % CO 2 배양기에서 37 O C에서 배양 플레이트를 품어. </li><li> 현미경 검사에 의해 매일 세포의 성장을 모니터링합니다. 문화의 7 일 (그림 3.A) - 증식하는 세포의 작은 클러스터는 5 후 현미경으로 관찰 할 수있다. NOTE :이 시점에서 T 세포 배양의 시각화가 어려울 수 있으며,이 단계에서 관찰 가능한 셀 클러스터의 부재가 성장하는 T 세포의 결여를 지시하지 않을 수있다. </li><li> 세포 격리 후 십사일에서 조심스럽게 문화의 상단 오프 미디어 100 μl를 제거하여 피드 문화 100 μ로 교체고 l 신선한 인간 T 세포 배양 배지. 6 % CO 2 배양기에서 37 O C에서 배양 플레이트를 배양 계속합니다. </li><li> 현미경 검사 및 배지의 색의 평가에 의해 매일 세포 성장을 모니터링. 3 일 매체 변경 후 - 대형 전지 클러스터는 현미경 2 볼 수 있어야합니다. 거시적으로, 세포 펠렛은 매체 변화에 따른 14 일 동안 볼 수있게해야합니다. </li><li> 세포 격리를 다음 이십팔일에서 현미경 검사로 성장하는 클론을 식별합니다. 200 μl의 인간 T 세포 배양 배지를 함유하는 48 웰 조직 배양 플레이트의 개별 웰에 성장 양성 96- 웰 플레이트의 배양을 200㎕ 부피를 전송합니다. </li><li> 항 CD3 자극 / 항 CD28 마이크로 비드 12.5 μl를 함유하는 100 μl를 추가 (- 5.6 섹션 5.2에 기재된 바와 같이 제조 됨). 6 % CO 2 배양기에서 37 O C에서 배양 플레이트를 품어. </li><li> 현미경 전 매일 문화의 성장을 모니터링민화 및 배지의 색의 평가. T 세포 배양은&gt; 2 × 6 / ㎖에 도달 할 때 (일반적 3-5일 재 자극 후, 미디어 시작되는 날에 의해 추정 될 수 지푸라기 황변)를 24- 웰 조직 배양 플레이트의 웰에, 전사 문화 500 μl의 인간 T 세포 배지를 추가한다. </li><li> 10~14일에서 pMHC 량체의 분석 바인딩 (섹션 3, 4.1에 기술 된 라벨 및 게이트 전략을 사용하고 있습니다) 유동 세포 계측법에 의해 alloantigen 특이성을 평가하기 위해 T 세포 배양 200 μl를 수집, 재 자극 따릅니다. </li></ol> 6. 장기 다시 자극 및 문화 T 세포 클론의 <ol><li> 지속적으로 14 일마다 할 수 있습니다 원하는 특이 클론 T 세포 배양을 다시 자극하고 확장합니다. 마지막 항 CD3 / CD28의 마이크로 비드 자극 다음 14 일에서 멸균 5 ML 튜브에 T 세포 배양를 수집합니다. 2.5 ml의 부피로, 하나의 튜브에서 동일한 T 세포 클론의 여러 웰을 결합. </li&gt; <li> 2.5 ml의에 최종 볼륨을 가져오고 피펫으로 부드럽게 혼합 매체를 추가합니다. </li><li> 배양 물로부터 이전 마이크로 비드를 제거하기 위해 세포 분리 자석으로 T​​ 세포 배양을 포함하는 5 ㎖의 튜브를 놓는다. 캡 튜브 위에 느슨하게해야한다. 실온에서 5 분 동안 인큐베이션. </li><li> 부드럽게 튜브의 캡을 제거합니다. 함께 튜브와 자석을 잡고, 신선한 5 ML 튜브에 세포 현탁액을 가만히 따르다. 누르거나 액체의 마지막 방울을 제거하기 위해 튜브의 내용을 흔들지 마십시오. </li><li> 4 ml의 최종 볼륨을 가지고 매체를 추가합니다. 샘플 10 μL은 혈구를 사용하여 세포를 계산합니다. </li><li> 실온에서 5 분 동안 600 XG에서 원심 분리하여 T 세포 복구. </li><li> 가만히 따르다 뜨는. 10 106 세포 / ml의 인간 T 세포 배양 배지에 재현 탁 T 세포. 전송 한 24 웰 조직 배양 플레이트의 각 웰에 분취 량. </li><li> 자극 항 CD3 / 12.5 μl를 함유하는 100 ㎕의 미디어를 추가하여 T 세포를 자극 재NTI-CD28 마이크로 비드 (- 5.6로 섹션 5.2 참조). 6 % CO 2 배양기에서 37 O C에서 품어. </li></ol>

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+estimation+of+the+human+&#945;&#946;+T+cell+receptor+diversity.">A direct estimation of the human &#945;&#946; T cell receptor diversity.</a> Science. 286 (5441), 958-961 (1999).</li><li>Robbins, H. 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=Comprehensive+assessment+of+T-cell+receptor+&#946;-chain+diversity+in+&#945;&#946;+T+cells.">Comprehensive assessment of T-cell receptor &#946;-chain diversity in &#945;&#946; T cells.</a> Blood. 114 (19), 4099-4107 (2009).</li><li>Alanio, C., Lemaitre, F., Law, H. K. W., Hasan, M., Albert, M. 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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

T 림프구는 적응 면역 시스템의 중요한 구성 요소입니다. T 세포뿐만 아니라 직접 효과기 메카니즘을 통해 다양한 병원균에 보호 면역 반응을 매개 할뿐만 아니라 적극적으로 자기 면역 관용을 유지하고 면역 체계에서 다른 세포의 반응을 지시 할 책임이있다. 이러한 함수는 T 세포 수용체 (TCR) 결찰, 사이토 카인 및 케모카인, 및 대사 산물 일 포함한 집적 신호의 숫자를 통해 지향된다. 이 적응 면역으로 T 세포의 역할을 정의하는 특성 특이성을 제공하기 때문에 이러한 신호 중, TCR은 특히 중요하다. TCR은 T 세포의 이펙터 기능을 시작하는 신호를 제공하기 위해 민감도와 특이도가 매우 높은 방식으로 MHC (인간의 ortholog의 HLA) 분자 (pMHC 단지)에 의해 제시 선형 펩티드 항원과 상호 작용한다. pMHC 리간드와 TCR 상호 작용의 생화학 매개 변수 T에 대한 특이성뿐만 아니라 제공세포 활성화는 또한 후속하는 T 세포의 기능 (2)에 정 성적 영향을 미친다. 따라서 공부 T 세포의 기능은 종종 정의 항원 특이성 T 세포 클론의 응답을 검사하는 요구한다. 10 8 별개 αβTCRs 3-4 - 약 10 12 αβ의 T 세포를 함유하는 인간 T 세포 구획, 추정 된 107를 포함한다. 이 다양한 레퍼토리 보호 면역에 대한 T 세포 반응을 필요로 할 가능성이 병원균에서 펩타이드의 광대 한 배열의 인식을위한 기회를 제공한다. 그 항원 5 종래 면역 반응의 부재 10-7 - 이는 자기 MHC 제시 주어진 외래 항원에 대한 T 세포 응답의 주파수는 10-4 정도이다 것으로 추정된다. 나이브 T 세포 레퍼토리는 펩티드 항원 및 한계 반응 제시 자기 MHC 인식하는 능력을 보장하기 위해 흉선 선택에 의해 형성된다자기 항원에 대한 펩타이드의 ivity 보호 면역성이 중개 용 잠재 성을 극대화. 이 위반 반응성, 비교적 큰 주파수, 10-3 설계에서 그러나, - 면역 나이브 개체로부터 T 세포 10-4을, 외국인 MHC 분자뿐만 아니라 그들이 제시 내인성 펩티드를 모두 인식 동종 세포 자극에 반응 6. 동종 pMHC 리간드의 인식은 자기 MHC 제시 외국 항원의 인식과 구조적으로 유사하다; TCR은 동종 MHC 분자뿐만 아니라 펩티드 제시 7 모두 중요한 생화학 적 상호 작용을한다. pMHC의 다양성으로부터 동종 자극의 결과로 T 세포 반응의 강력한 특성상 동종 세포 (8)의 표면에 존재하는 복합체. 이것은 각각의 MHC 약 2 × 4 다른 내인성 펩티드 항원 제시 9 것으로 추정된다. 이 B동종 자극에 대한 응답의 readth은 T 세포 alloreactivity 인한, 이러한 숙주 질환 (GVHD) 대 동종 이식 거부 반응 또는 이식편과 같은 임상 적으로 관련 병리학의 중요한 측면이다. 인간 T 세포 alloreactive 반응 연구는 전통적으로 동종 세포 자극 이후 나이브 T 세포의 클론 반응 검사에 의존하고있다. 한계 희석 조합 같은 동종 세포주 반복 자극은 동종의 HLA (10)의 정의와 인식 클론 T 세포를 생성 할 수있다 분석한다. 내인성 pMHC의 크고 다양한 HLA 레퍼토리는 T 세포의 광범위한 레퍼토리를 자극 주어​​진 동종위한 본 복합체 그러나,이 접근법은, 개별 pMHC 동종 리간드에 대한 반응을 검사하는 문제가있다. 이 벌크 인구 자극 제한 희석 방법은 원하는를 Reacti와 T 세포를 분리하기 위해 다수의 클론의 스크리닝을 필요하나의 pMHC 리간드에 대한 vity. 또한, 개별 pMHC 동종 리간드에 반응 T 세포의 빈도는 소정의 항원에 응답하여 인간 T 세포 클론의 효율적인 생성에 대한 장벽을 제공 나이브 T 세포 집단 중에서 비교적 낮다. 식별 및 클론 집단에서 항원 특이 적 T 세포의 분리는 형광 표지 된 pMHC의 다량 체 (11)의 현상에 의해 활성화되었습니다. 이 접근 방법은 스트렙 타비 딘 - 표지 된 형광 물질에 결합함으로써 표시되어 재조합 가용성 바이오틴 MHC 분자에로드 특정 펩타이드 항원을 이용한다. pMHC의 Multimerization 수용성 pMHC 리간드 TCR의 본질적 낮은 (μM)의 친 화성을 보상 결합력을 증가시킨다. 표지 된 세포를 식별하고 유동 세포 계측법에 의해 단리 할 수​​있다. 그러나,이 접근은 아직 일반적이다 나이브 T 세포 집단 중 항원 특이 적 T 세포의 낮은 주파수에 의해 제한된다대부분의 유동 세포 계측기에 대한 정확한 확인 및 정량 한계 이하의 크기 순서. 이러한 한계를 해결하기 위해, 테트라 - 표지 된 세포 pMHC 량체 라벨 및 후속 자성 비드 농축 방법 (12)을 개발되었다. 이 방법은 신뢰할 수있는 검출, 열거 및 저주파수 항원 특이 적 T 세포의 분리를 보여 주었다. 이 프로토콜은 특별히 개별 pMHC 동종 리간드에 반응 인간 T 세포 클론의 생성을위한 유효 프로토콜을 설명한다. 프로토콜은 단일 정렬 세포로부터 T 세포 클론의 생산 (활성화 세포 분류 유세포 인간 T 세포의 생체 외 배양을위한 견고한 방법 alloantigen 특정 인간 T 세포의 분리를 위해 합체 라벨링 및 농축 pMHC (HLA)을 적용 그림 1의 개요).

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Sodium heparin venous blood collection tube 16 x 100 mm</td>
			<td style="width:223px;">Becton, Dickenson and Company</td>
			<td style="width:119px;">366480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Lymphoprep</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">7801</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:273px;">Rosette Sep Human T Cell Enrichment Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">15061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dulbecco&#39;s PBS, 1x without Ca or Mg</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fluorophore-labeled pMHC tetramer</td>
			<td style="width:223px;">NIH Tetramer Facility</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Biotin Selection Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Selection magnet</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:273px;">TruStain FcX Human Fc blocking solution</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">422301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD5 PE-Cy7 (clone UCHT2)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">300621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD14 FITC (clone HCD14)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">325603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD19 FITC (clone HIB19)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">302205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Iscove&#39;s DMEM, without b-ME or L-glutamine</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">15-016-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin sulfate (50 mg/ml)</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">GT-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human AB serum, male donor</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">HS-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2</td>
			<td style="width:223px;">Peprotech</td>
			<td style="width:119px;">AF 200-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Dynabeads Human T-Activator CD3/CD28</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">11131D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Media</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cell sorting buffer</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS, pH 7.4</td>
			<td style="width:223px;">1 L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BSA</td>
			<td style="width:223px;">10g</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA (0.5 M)</td>
			<td style="width:223px;">2 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human T Cell Culture Medium</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Iscove&#39;s DMEM</td>
			<td style="width:223px;">351.6 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Heat-inactivated human AB serum</td>
			<td style="width:223px;">40 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES (1 M)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax (100 x)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin (50 mg/ml)</td>
			<td style="width:223px;">0.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-mercaptoethanol (14.3 M)</td>
			<td style="width:223px;">1.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2 (1 mg/ml)</td>
			<td style="width:223px;">1 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

전략 정렬 계측법 자성 비드 농축 및 단일 셀을 통해 유동 특이성 alloantigen 정의와이 프로토콜은 클론 인간 T 세포 배양의 생성을 설명한다. (1)가 처리의 개요를 제공하는 도표. <img alt="그림 1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> 그림 1 : 프로토콜 개요 프로토콜은 여기에 설명 된 말초 혈액에서 alloant...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K 조회수.  University of California, San Diego. 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.

비디오: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

연구

면역학 및 감염병학

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

공동 stimulatory 신호 봉쇄와 Alloantigen 자극에 의​​해 사람 말초혈 Mononuclear 세포 Alloantigen 특정 Anergy의 유도

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Zoledronate를 사용하는 사람 말초혈의 확장 γδ T 세포

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

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 ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

임상 신청<em&gt; 잠자는 숲속의 미녀</em유전자 주변과 배꼽 탯줄 혈액에서 T 세포를 수정하는 셀을 제시&gt;과 인공 항원

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

전혈로부터 인간 단핵구 유래 수지상 세포의 생성

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

희귀 한 항 원 특정 B 세포 혈액 순환에서 구별 인간 단일 클론 항 체의 생성

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

말 초 혈액 및 림프 조직에서 기본 인간 T 세포의 시 냅 스 인터페이스의 평가

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

부유 기반 T 세포 분리, 활성화 및 마이크로버블을 사용한 인간 말초 혈액 단핵 세포 샘플로부터의 확장

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

기능성 파골세포와 인간 말초 혈액 CD14+ 단핵구의 분화

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...