We thank Joon Kim for expert flow cytometry assistance. Research in the authors&#8217; labs is supported by the SCORE program of the National Institutes of Health (grant SC1-GM095402 to B.D.O) and the Canadian Institutes of Health Research (to J.C.Z.P.). J.C.Z.P. is supported by a Canada Research Chair in Developmental Immunology. The biomedical research infrastructure of Hunter College is supported in part by the NIH Research Centers in Minority Institutions (RCMI) program via grant MD007599. We also acknowledge the New York State Stem Cell Science Program (NYSTEM) for its support of the initiation of stem cell research at Hunter College via grant C023048.

문화 미디어, 사이토 카인 및 젤라틴 판의 1 준비 <ol><li> 높은 포도당과 피루브산 나트륨과 이글의 중간 (DMEM) 둘 베코의 변형 례를 사용하여 ES 세포 매체를 준비한다. 1 % HEPES 버퍼, 1 % 비 필수 아미노산, 0.1 % 겐타 마이신 (50 ㎎ / ㎖) 및 0.1 % (태아 혈청 (FBS)를 공인 20 % ESC, 1 % 페니실린 / 스트렙토 마이신, 1 % L-글루타민을 추가 55 μM) β-머 캅토 에탄올. 여과에 의해 ES 세포를 멸균 매체. </li><li> 제조업체의 지침에 따라 분말 알파 최소 필수 중간 (α-MEM)의 1 L함으로써 OP9 미디어를 준비합니다. 20 % 소 태아 혈청 (FBS), 1 % 페니실린 / 스트렙토 마이신을 추가합니다. 혼합하는 반전. 이 500 ㎖의 분취 액으로 여과하여 멸균. 참고 : 분말로 만든 미디어의 사용은 권장 개선 OP9 세포의 유지 보수 및 시험 관내 분화 결과를 얻을 가능성이있다. 이 방법은 우리의 표준 절차가되었다. 그러나, 우리는 그쪽에주의t 우리는 시간에 적절한 성공을 미리 만들어진 액체 α-MEM을 사용했다. </li><li> 90 % FBS 10 % 디메틸 설폭 사이드 (DMSO)를 추가하여 미디어를 동결 준비합니다. 부드럽게 소용돌이 여과 소독. 4 ℃에서 보관하십시오. </li><li> 10 μg / ml의 완전한 OP9 미디어에서 인간 재조합 FLT-3 리간드 (FLT-3L)을 용해하여 2,000x FLT-3 리간드 (10 μg / ㎖)을 준비합니다. -80 ° C에서 1.5 ML의 microcentrifuge 튜브로 나누어지는 및 저장. 안정성 및 저장에 대한 공급 업체의 권장 사항을 따르십시오. 참고 : FLT-3L의 안정성 (4 ° C 또는 3개월 -80 ° C에서의 1 개월) 짧습니다. </li><li> 전체 OP9 미디어를 사용하여 1,000 배 IL-7 (1 μg / ㎖)를 준비합니다. -80 ° C에서 1.5 ML의 microcentrifuge 튜브로 나누어지는 및 저장. 안정성 및 저장에 대한 공급 업체의 권장 사항을 따르십시오 (IL-7은 -80 ° C에서 최대 12 개월 동안 안정적이다). </li><li> LIF의 난의 열 일곱 단위의 1:10 희석 1,000 배 백혈병 억제 인자 (LIF) (10 μg / ㎖)를 준비N ES 세포 매체. 4 ℃에서 보관 나누어지는. 나누어지는 병에 제조업체가 제공하는 유효 기간을주의해야합니다. 참고 : 제품이 제조 일로부터 농축 또는 희석 된 형태로 적어도 18 개월 동안 안정적이다. </li><li> 젤라틴 6 - 웰 플레이트를 준비하여 6 - 웰 플레이트의 각 웰에 1.5 ml의 0.1 % 젤라틴 용액을 추가한다. 코팅에 적어도 30 분 수에 뚜껑 실온에서 멸균 후드에서 요리를 둡니다. 요리는 가습 인큐베이터 하룻밤에 남아있을 수 있습니다. 바로 세포 파종 전에 남은 젤라틴 용액을 제거합니다. 젤라틴 용액이 완전히 우물에 건조시키지 마십시오. </li></ol> 2 준비 및 피더 세포 및 마우스 배아 줄기 세포의 유지 보수 (MESC) 주 : 5 % CO 2 가습 37 ° C 배양기에서 모든 세포를 품어. <ol><li> 해동 마우스 배아 섬유 아세포 (MEF) <ol><li> 냉동 바이알 (~ 5 × 10 6 빠른 해동 </SUP&gt; 셀 / 마이 토마 이신 C 처리 (또는 다른 mitotically 체포) MEFs의 유리 병)하고 15 ML 튜브에 전송할 수 있습니다. </li><li> 점차적으로 동결 매체로부터 DMSO를 희석 드롭 현명한 방식으로, 해동 세포에 ES 세포 매체의 8 ML을 추가합니다. </li><li> 5 분 동안 4 ° C에서 400 X g에서 원심 분리기 세포. 조심스럽게 상층 액을 제거하고 ES 세포 미디어 3 ㎖에서 세포 펠렛을 재현 탁. </li><li> 미리 예열 ES 세포의 미디어 (33)를 가하여 50 ㎖의 원심 분리 관을 준비한다. 36 ml의 최종 볼륨을 가지고 재 부유 MEFs의 3 ML을 추가합니다. </li><li> 이 젤라틴 6 웰 플레이트의 각 웰에 재 부유 MEFs 3 ㎖를 배포합니다. 세포가 부착하고 그들에 mESCs 시드 전에 확산 할 수 있도록 최소 6 시간 동안 인큐베이터에 접시를 놓습니다. 자신의 미디어를 2-3 일마다 변경되는 경우 이러한 mitotically 체포 피더 층은 초기 시딩 후 몇 일 동안 사용할 수있다. 주 : 갓 체포 MEFs도 사용할 수 있습니다. </li> &lt;/ OL&gt; </li><li> 시드 mESCs <ol><li> 37 ° C의 물을 욕조에 얼어 붙은 MESC 클론 유리 병을 빠른 해동 2.1.1-2.1.3에 설명 된대로 세포를 준비합니다. 참고 : 우리는 6 웰 플레이트의 합류 우물에서 MESC의 두 병을 동결. </li><li> (단계 2.1.5에서 준비) 체포 MEF 단일 층과 6 - 웰 플레이트의 (MESC 클론 당) 한 우물에서 용지를 제거하고 MEF 단일 층의 상부에 mESCs의 3 ml의 씨앗. 1,000 배 LIF (10 NG / ㎖)의 3 μl를 추가합니다. 인큐베이터에 요리를 돌려줍니다. </li></ol></li><li> ES 세포 유지 보수 및 트립신 매개 통로 참고 : MESC의 합류에 따라 변경 매일 미디어, 또는 분할. 최적의 수확 및 분할 / 재 판 세포 매일. <ol><li> ES 세포 미디어를 제거하고 PBS의 2 ㎖로 mESCs을 씻는다. PBS를 제거합니다. 0.25 % 트립신의 1 mL를 넣고 3 ~ 5 분 동안 37 ° C에서 배양한다. </li><li> 트립신 세포를 수집하고 15 ㎖의 원심 분리기 튜브로 전송할 수 있습니다. 적극적 m의 덩어리를 분리시킨 세포 현탁액을 피펫ESCS. 트립신을 중화하기 위해 세포 현탁액에 완전한 ES 세포 미디어 2 ML을 추가합니다. </li><li> 4 ° C에서 5 분 동안 400 X g에서 스핀 세포. 상층 액을 제거하고 3 ML의 ES 세포 미디어에서 펠렛을 재현 탁. </li><li> 준비 체포 MEF 단일 층을 포함하는 6 - 웰 플레이트에서 용지를 제거합니다. 각 웰에 ES 세포의 미디어 3 ㎖ 중의 (원하는 분할 비에 기초하여) 세포 현탁액의 적절한 양을 추가하는 시드된다. 1,000 배의 LIF의 3 μl를 추가합니다. 이일에 통과 할 수있는 상태가됩니다 6 : 합류 MESC 잘 일을 분할합니다. </li></ol></li><li> OP9 / OP9-DL1 세포 유지 <ol><li> OP9 세포의 유리 병을 해동 (단계를 수행 2.1.1-2.1.3 OP9 미디어를 사용) </li><li> 10cm 조직 배양 접시에 7 ML의 OP9 미디어를 추가합니다. 플레이트를 통해 세포를 배포, 드롭 현명한 방식으로 재현 탁 세포의 3 ML을 추가합니다. 배양기에서 하룻밤 세포를 놓습니다. </li><li> 다음날 OP9 세포의 합류를 확인합니다. 요리는 많은 죽은 부동 세포를 포함하는 경우, 레모미디어를했습니다 신선한 OP9 미디어 10 ㎖를 추가합니다. 거의 완전 합류가 관찰되지 않은 경우 인큐베이터에 접시를 돌려줍니다. 네 근처에 합류 OP9 단층 : 분할 1 (트립신을 사용하여). 주 : 플레이트에 대한 이일에 다시 합류 동일한 수준에 도달해야한다. 오버 합류하게 OP9 세포를지지 않도록주의 해주십시오. </li><li> 확장 주식으로 OP9 세포의 조기 구절을 동결. 참고 : 우리가 합류 10cm 접시 근처에 하나에서 OP9 세포의 두 병을 동결. 확장 주식의 각각의 바이알에 대해서는 나중에 더 MESC 공동 문화 실험에 사용되는 작업 주식을 만들 전파 될 수있다. 온도 변동에 부정적인 정지의 품질에 영향을 미칠 수 있기 때문에, 오히려 -80 ° C 냉동고에 비해 액체 질소에 냉동 모든 OP9의 주식을 유지합니다. </li></ol></li></ol> 3 OP9-DL1 공동 배양 절차 <ol><li> 공동 배양 준비 <ol><li> 약 일주 전에 공동 문화의 시작에, MEFs, mESCs 및 OP9 전지 작업 STO 해동CKS. OP9-DL1 세포를 공동 배양 일 8까지 필요하지 않기 때문에, 공동 문화 개시 후 처음 삼일 동안 OP9-DL1 세포를 해동. 유지 또는 필요에 따라 모든 셀을 동결. </li><li> 실험 규모에 기초하여 일 0 공존 배양에서 80 % 합류점에 도달 제조 OP9 세포 단층을 가진 10cm 플레이트의 초기 수를 결정 (예 MESC 클론의 수는 차별화되는 공존 배양 시점의 개수 ) 분석. 4 일 : 공동 문화를 준비하기 위해 한 사이 OP9 세포의 합류 접시 근처에 분할 6 이일을 앞두고 단일 층이 필요합니다 때의. 참고 : 하나는 ESC 클론 당 적어도 하나의 플레이트가 필요합니다. 5 일 경과 6 개의 판을 씨앗에 충분한 세포를 산출한다 0 일에 (아래 설명 참조) 일반적으로, ESC 단일 클론은 단일 플레이트에 시딩. 매일 5 판은 하나의 후속 분석 시점을 가능하게 할 것이다. </li><li> OP9 단일 층이 지속적으로 공동 문화의 통과를 위해 필요한 것입니다 때문에,주의 항상 터 유지 보수에 걸릴공동 문화와 병렬로 추가 OP9 배양 플레이트의 적절한 수있다. </li></ol></li><li> 일 0 : 공동 문화의 개시 <ol><li> 2.3.1-2.3.4에서와 같이 MESC를 수집합니다. 세포를 계산합니다. </li><li> 각 MESC 클론은 접시 당 OP9 미디어 10ml에 5 × 10 4 MESC를 준비합니다. NOTE :. 우리가 사용되지 않았지만, 우리는 대안 매체는 α-MEM, 10 % FBS로 이루어진, 5 × 10 β-머 캅토 에탄올이 또한 분화의 공동 배양에 사용하기 위해보고되었다 -5 M 10 참고 </li><li> OP9 요리에서 이전 용지를 제거하고 접시에 골고루 세포를 배포 돌보는 요리에 MESC 현탁액의 10 ML을 추가합니다. 37 ° C 배양기에서 접시를 놓습니다. </li></ol></li><li> 주 3 : 미디어 변경 <ol><li> 인큐베이터에서 요리를 제거하고 현미경으로 관찰한다. 콜로니의 광택을 잃게하고 평평하게 나타납니다 시작해야한다. </li><li> 요리에서 용지를 제거하고 부드럽게 신선한 OP9 10 ㎖를 추가미디어. </li><li> 인큐베이터에 공동 배양 접시를 돌려줍니다. 시각 콜로니 80-90 %의 시각적 중배엽 같은 형태를 표시 ~까지 수행되지 않아야 다음 통로 (5 일)에 대한 OP9 피더 단층 제제의 타이밍을 알리는 일일 중배엽 형 콜로니 형성을 모니터. 5 일째 세포 전사 공정의 최대 연기 2 일이다. </li></ol></li><li> 제 5 일 : 사전 도금 트립신 매개 통로입니다. 참고 : 최적의 합류 OP9 세포를 사전에 10cm 요리의 적절한 수를 준비합니다. 공동 문화 시각적 단계 3.3.3에 설명 된 기능을 표시하는 경우에만 다음 단계를 진행합니다 (또한 대표 결과 참조). <ol><li> 공동 배양 접시에서 미디어를 꺼내십시오. 씻어 4 ml의 PBS를 추가합니다. PBS 소용돌이 및 제거합니다. 0.25 % 트립신의 4 mL를 넣고 ~ 5 분 동안 인큐베이터에서 접시를 놓습니다. </li><li> 돌보는 것은까지 과도한 기포를 도입하지 격렬한 피펫 팅함으로써 세포를 트립신의 층을 방해균질 주로 단일 세포 현탁액을 얻을 수있다. </li><li> 전체 OP9 미디어의 4 mL를 넣고 피펫으로 혼합한다. 30 분 동안 인큐베이터에서 비어있는 새 10cm 접시와 장소에 전송 세포 OP9 세포가 접시에 부착 할 수 있도록합니다. 이러한 &quot;도금 전&quot;단계는 공동 배양의 다음 단계로 이송 OP9 세포의 수를 감소시킨다. </li><li> 40 μm의 셀 스트레이너 50 ㎖의 원심 분리기 튜브를 준비합니다. </li><li> 사전 도금 접시에서 비 부착 세포를 수집하고 튜브에 세포 스트레이너를 통해 전달합니다. PBS의 6 ㎖로 가볍게 요리를 세척하고 뒤에 첨부 된 OP9 세포를 떠나, 같은 여과기를 통과. </li><li> 4 ° C에서 400 × g으로 5 분 원심 분리기 세포. 상층 액을 제거하고 완전한 OP9 미디어의 3 ㎖에 펠렛 세포를 재현 탁. 세포를 계산합니다. </li><li> 최적의 합류 OP9 세포 10cm 플레이트에서 용지를 제거합니다. </li><li> 각 분석 시점 들어 OP9 모노에 5 × 105 세포를 시드10 ㎖의 최종 부피의 층을 포함한다. </li><li> 5 NG / ML 인간 재조합 FLT-3L를 추가하고 배양기에서 접시를 놓습니다. </li></ol></li><li> 8 일 : 조혈 전구 세포 (HPC) 컬렉션 참고 :이 단계를위한 시간들이 될 수 있도록 사전에 OP9 또는 OP9-DL1 세포 단일 층과 6 - 웰 플레이트의 적절한 수를 준비 ~ 80 %의 합류. <ol><li> 40 μm의 여과기와 50 ㎖의 원심 분리기 튜브를 준비합니다. </li><li> 인큐베이터에서 요리를 제거하고 현미경으로 관찰한다. HPC의 반짝이 클러스터는 느슨하게 OP9 단층에 연결해야합니다. 피펫 OP9 단층과 접시에 기존 미디어를 세척 (그러나 지나치게 방해하지 않음)에 의해 가능한 한이 세포의만큼을 수집합니다. 거품을 방지하기 위해, 항상이 HPC 수집 / 세척 단계에서 피펫의 끝에서 용지의 최소 1 ml에 둡니다. </li><li> 튜브에 40 μm의 스트레이너를 통해 세포를 전달합니다. 만약 모든 HPC의 동일 판에 8 ㎖의 PBS를 추가하고 현미경으로 확인 우리다시 수집. 더 강력한 세척으로 (필요한 경우) PBS로 세척 / 수집 단계를 반복합니다. 이미 동일한 플레이트에서 세포를 함유하는 튜브에 세포 스트레인. </li><li> 4 ° C에서 5 분 동안 400 × g으로 원심 분리기 세포. </li><li> 5 NG와 OP9 미디어 (판은 5 일에 시드 당) 3 ML의 마스터 믹스를 준비 / ㎖ FLT-3L 1 NG / ml의 IL-7. </li><li> FLT-3L + IL-7 마스터 믹스 OP9 미디어에서 (처리 된 각 10cm 접시 3 ml)에 뜨는에 resuspend 펠렛을 제거합니다. OP9 세포와 6 웰 플레이트의 한 우물에 각 판에서 수집 된 세포를 전송합니다. <ol><li> , 단구, B 세포 계통 또는 적혈구 세포를 향해 이용시 OP9 세포 상에 수집 된 세포를 시드하기 위해. T 세포를 유도하기 위해, OP9-DL1 세포 단층 상에 세포를 시드. </li></ol></li><li> 인큐베이터에 6 - 웰 플레이트를 놓습니다. </li></ol></li><li> 10 일 : 미디어 변경 <ol><li> 각각 별개 MESC 클론 피더 셀형 combina 대해 15 ml의 원심 분리 튜브를 준비공동 문화 기. </li><li> FLT-3L의 5 겨 / ㎖와 IL-7의 1 겨 / ㎖로 OP9 미디어의 마스터 믹스를 준비합니다. 각 웰에 3 ML을 준비합니다. </li><li> 부드럽게 같은 ESC 복제 피더 세포 유형의 조합을 포함하는 여러 우물에서 미디어를 결합 6 웰 플레이트의 각 웰에서 미디어를 수집합니다. </li><li> 각 웰에 OP9 미디어 마스터 믹스 2 ㎖를 (3.6.2 참조)에 추가합니다. </li><li> 원심 분리기 4 ° C에서 5 분 동안 400 X g에서 수집 된 미디어. 물론 당 OP9 미디어 마스터 믹스 1 ㎖의 각 펠렛 (볼 경우 매우 작은) (3.6.2 참조) 원래 단계 3.6.3에서 수집을 상층 액을 제거하고 재현 탁. </li><li> 다시 각 웰 미디어가 원래 3 ml의 각 웰에 볼륨을 가져 수집되는 세포에 1 ㎖를 배포합니다. 인큐베이터에 접시를 돌려줍니다. </li></ol></li><li> 주 12 : 아니 트립신 통로 참고 :이 초 동안 그들은 최적의 시간에 컨 플루 언트 될 수 있도록 사전에 OP9 또는 OP9-DL1 세포를 6 웰 요리의 적절한 수의 준비스탭. 날 (12)은 현상 적혈구, 단핵구, 및 초기 단계의 T 세포의 유세포 분석에 적합하다. <ol><li> 마스터 믹스 FLT-3L의 5 겨 / ㎖와 IL-7의 1 겨 / ㎖로 OP9 미디어 (공동 문화에 계속 각 웰 3 ㎖) 준비합니다. </li><li> 40 μm의 여과기 (공동 문화의 각 ESC 복제 피더 세포 유형의 조합 일)과 50 ㎖의 원심 분리기 튜브를 준비합니다. </li><li> 적극적으로 잘에 (단일 층을 포함하여) 모든 세포를 해리하는 각도에있는 기존 미디어를 피펫. 단일 세포 현탁액을 닮은 상태가 될 때까지 강력한 피펫 계속합니다. </li><li> 튜브에 스트레이너를 통해 수집 된 세포를 전달합니다. 남아있는 모든 세포를 씻어 수집하기 위해 각 웰에 PBS의 3 ML을 추가합니다. 같은 스트레이너를 통해 세포를 전달합니다. 동일한 클론 ESC 피더 세포 유형 조합을 포함하는 다수의 웰에서 세포 수확기. </li><li> 사용 된 셀 스트레이너를 취소하고 잘 하나에 나누어 상당을 제거 (~ 6 ml)을 원하는에 대한(또는 다른) 유동 세포 계측법은 일에 실시되는 분석한다. 사용하기 전에 얼음이 분주을 저장합니다. </li><li> 4 ° C에서 5 분 동안 400 × g으로 원심 분리기 세포. 상층 액을 제거합니다. 물론 당 마스터 믹스의 3 ㎖에 50 ㎖의 원심 분리기 튜브에서 펠렛을 재현 탁은 다시 시드합니다. 적절한 피더 세포 유형에 각 세포 현탁액을 잘 당 3 mL를 넣고 배양기에서 접시를 놓습니다. </li></ol></li><li> 주 14 : 미디어 변경 <ol><li> 3.6 절에 설명 된 단계를 따릅니다. </li></ol></li><li> 주 16 : 아니 트립신 통로 참고 :이 단계에서 사전에 최적으로 합류 OP9 또는 OP9-DL1 세포를 6 웰 요리를 준비합니다. 참고 : 유동 세포 계측법 B 림프구 및 중간 단계의 T 세포 분석을위한 날 (16)가 이상적입니다. <ol><li> 3.7 절에 설명 된 단계를 따릅니다. </li></ol></li><li> 주 18 : 미디어 변경 <ol><li> 3.6 절에 설명 된 단계를 따릅니다. </li></ol></li><li> 주 20 : 아니 트립신 통로 주 : 일 20 전유동 세포 계측법에 대한의 이상적인는 T 세포를 개발 최종 단계에 중간 분석. <ol><li> 3.7 절에 설명 된 단계를 따릅니다. 원하는 경우, 세포에게 과거의 일 (20) 교류 매체의 변화를 차별화 수행하고 더 트립신은 림프구의 확장 속도를 매일 모니터링, 모든 이일 통로 없습니다. </li></ol></li></ol>

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The authors have nothing to disclose.

OP9-DL1 공동 배양 시스템은 줄기 세포에서 혈액 세포 유형의 개발시 각종 유전자 산물의 역할을 연구하기 위해 이용되어왔다. 8,12,13 또한 유전자 조절 DNA의 기능을 연구하기위한 효과적인 모델을 입증 세포 분화 과정. 9,14 시간 및 조혈 많은 기본적인 질문을 해결 실험에서 상당한 비용 절감 효과를 얻을 수있는 전체적인 마우스 모델의 대안으로이 방법을 사용. 그러나 이러한 목적...

A cell culture system has been established in which mouse embryonic stem cells (mESC) are differentiated to T cells in vitro.1 This system exploits the ability of Notch signaling to drive T cell differentiation.2 The OP9-DL1 cell line was created by transducing bone marrow-derived OP9 cells3 with a Notch ligand, Delta-like 1 (DL1).4 Activation of the Notch signaling cascade in vitro facilitates T cell development to the exclusion of other cell lineages. With the inclusion of appropriate cytokines, this system provides a cell culture &#8220;microenvironment&#8221; that supports the sequential advancement of mESC toward hematopoietic and ultimately T cell lineages. This system supports the flow cytometric identification of T cells at the various developmental stages seen during normal T cell ontogeny in the thymus. For investigating selected questions relating to T cell development, this procedure has become an attractive alternative to in vivo whole mouse models5 and in vitro fetal thymic organ culture methods used to elicit T cell development from mouse embryonic stem cell derived hematopoietic precursors.6 The major advantage of the OP9 co-culture system is that it involves standard and straightforward cell culture techniques and does not depend on the continual use of experimental animals. 
We follow a detailed, previously published protocol in our experiments using this approach.7 We have utilized this technology to examine the hematopoietic differentiation products of non-manipulated mESC clones, high quality mESC clones handpicked to make chimeric embryos8 and stably-transfected ESC clones coming directly out of drug selection.9 We have noted that the temporal kinetics of initial in vitro differentiation from mESC to mesoderm-like colonies in this model can be variable among individual clones. The mESC-OP9 co-cultures can be visually assessed for progression to mesoderm. While this will usually be completed by the fifth day of co-culture, among individual clones, completion can be delayed for one or two days. Quantitative (~80-90%) mesoderm formation must be achieved prior to transfer in order to obtain optimal hematopoietic progenitor cell (HPC) formation and robust lymphopoiesis. Thus, when working with multiple mESC clones, this &#8220;day 5&#8221; passage is best delayed until all clones complete the transition to mesoderm-like colonies. This enables synchrony of subsequent development among the clones after their transfer into hematopoietic differentiation conditions. Three days after the passaging of the 80-90% mesodermal formations, HPCs are collected from the OP9 monolayers. HPCs can be seeded on new OP9 cells to allow differentiation of monocytic, erythroid and B cell lineages. Alternatively, HPCs can be seeded on OP9-DL1 cells and driven towards T cell development. All in vitro differentiation cultures are provided Flt-3L beginning at day 5, with further addition of IL-7 beginning at day 8. Flow cytometry analyses performed at various time points during the experiment enable monitoring of progress through the stages and lineages of hematopoietic differentiation and T cell development. CD4/CD8 double positive (DP) T cells begin emerging by day 16 of the co-culture, and both DP and CD8 single positive (SP) cells are abundant by day 20. The general outcome and robustness of co-culture is greatly dependent on the ability to visually ascertain the completion of the significant developmental turning points that occur. This protocol aims to be a guide to the recognition of these milestones, as well as the other critical parameters, that are key to successful differentiation.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:355px;">MATERIALS</td>
			<td style="width:127px;">COMPANY</td>
			<td style="width:141px;">CATALOG NUMBER</td>
			<td style="width:432px;">COMMENTS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Stem cell qualified FBS</td>
			<td>Gemini</td>
			<td>100-125</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin/Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-alanyl L-glutamine</td>
			<td>Corning</td>
			<td>25-015-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HEPES buffer&#160;</td>
			<td>Millipore</td>
			<td>TMS-003-C</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Non-Essential Amino Acids</td>
			<td>ThermoScientific</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gentamicin Regent Solution (50 mg/mL)</td>
			<td>Life Technologies&#160;</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#946;-mercaptoethanol (55 mM) in DPBS</td>
			<td>Life Technologies&#160;</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>0.22&#956;m PES membrane</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell Culture Grade Water</td>
			<td>Corning</td>
			<td>25-055-CM</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#945;-MEM&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>12000-022</td>
			<td>Powder, reconstitute per manufacture recommendation</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FBS</td>
			<td>ThermoScientific</td>
			<td>SH 30396.03</td>
			<td>Testing of individual lots required&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dimethyl Sulphoxide&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Human Flt-3 Ligand</td>
			<td>R&#38;D Systems</td>
			<td>308-FK</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Murine IL-7</td>
			<td>PeproTech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LIF&#160;</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Utrapure water with 0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MEFs mitomycin C treated</td>
			<td>Millipore</td>
			<td>PMEF-CF</td>
			<td>Any mitotically arresteded MEFs can be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DPBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypsin EDTA, 1X</td>
			<td>Corning</td>
			<td>25-053-Cl</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell strainer (40 &#956;m)</td>
			<td>Fisher</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue culture dish 100 X 20 mm</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiwell 6-well</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 ml microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>Tubes for FACS&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ES R1 cells</td>
			<td>ATCC</td>
			<td>SCRC-1011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9 cells</td>
			<td></td>
			<td></td>
			<td>Cells can be obtained from the Riken Laboratory Cell Repository (Japan).</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9-DL1 cells</td>
			<td>&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td>Cells can be requested from the Z&#250;&#241;iga-Pfl&#252;cker laboratory.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FlowJo software&#160;</td>
			<td>Tree Star</td>
			<td></td>
			<td>FACS data analyses</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Flow Cytometer</td>
			<td>BD</td>
			<td></td>
			<td>FACScan, FACSCalibur and FACSVantage have been used in our lab</td>
		</tr>
	</tbody>
</table>

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.

LIF의 존재 MEFs에 성장하면 mESCs는 미분화 상태로 유지 될 수있다. 이상적인 조건에서, 그들은 위상차 현미경에 빛나는 후광에 둘러싸인 세포의 소형 식민지 (그림 1)로 나타납니다. 이러한 문화는 매일 모니터링해야합니다. 세포의 합류에 따라 미디어가 변경 될 수 있거나 세포를 분리 할 수​​있다. 이웃 MESC 식민지는 서로의 접점에 와서는 안된다. 미분화 mESCs의 정상적이고 건강한 ?...

Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

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 ...

derivation-of-t-cells-in-vitro-from-mouse-embryonic-stem-cells

Research

JoVE Journal

Immunology and Infection

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

10.9K 조회수.  City University of New York. 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.

비디오: Derivation of T Cells In Vitro from Mouse Embryonic Stem 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

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

에서 Thymic 림프종 T - 세포 라인의 유도 ATM - / -</sup과 p53의 - / -</sup 마우스

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

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&#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 ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

T Lymphocytes쪽으로 유도 Pluripotent 줄기 세포의 분화 감독

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

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 ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

효율적인위한 킬러 인공 항원 제시 세포 (KaAPC)<em&gt; 체외</em&gt; 인간의 항원 특이 T 세포의 고갈

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

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 ...

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 ...

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.

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.