We thank Miltenyi Biotec, Germany for providing reagents and CliniMACS Prodigy equipment for evaluation studies. We thank George T. McNamara (Pediatric department, MD Anderson Cancer Center) for proof reading the manuscript. Grant support: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303; CA141303); R33 (CA116127); P01 (CA148600); Burroughs Wellcome Fund; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Estate of Noelan L. Bibler; Gillson Longenbaugh Foundation; Harry T. Mangurian, Jr., Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC&#8217;s Sister Institution Network Fund; Miller Foundation; Mr. Herb Simons; Mr. and Mrs. Joe H. Scales; Mr. Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; William Lawrence and Blanche Hughes Children's Foundation.

무균 조건에서 재료 1. 준비 (재료 및 장비 표 참조) <ol><li> 0.5 %의 최종 농도로 인간 혈청 알부민 (HSA)가 보충 된 PBS / EDTA 완충액을 제조 3 L (W / V). </li><li> 임상 등급 0.9 %의 염화나트륨 수용액 1 L 가방 및 GMP 급 세포 배양액 2 L을 준비한다. </li><li> 멸균 8 ml의 CMV pp65 유래의 한 유리 병을 재구성하여 CMV 특정 펩타이드 항원 칵테일의 60 nmol의를 준비합니다. </li><li> 루어 / 스파이크 인터 커넥터를 사용하여 50 ml의 볼륨 동결 가방에 거대 세포 바이러스 pp65 유래 펩티드 칵테일을 전송하고 튜브 세트에 칵테일 이후의 유통을 방지하기 위해 집게를 잠금으로 고정. 멸균 조건에서 열기 세포 농축 튜브 세트 (TS 500). </li><li> 멸균 튜브 용접기를 사용하여,이 시간에 펩티드 칵테일 가방 클램프를 열지 마십시오 튜브 세트의 밸브 2 TS (500)에 대한 튜브 연결에 펩티드 칵테일 냉동 가방을 연결합니다. </li><li> 제거 1 × 10 9 T셀룰러 출발 생성물로부터 NC 및 50 ㎖의 전체 부피로 2.5 % HSA를 함유하는 PBS / EDTA 완충액에서 중단. 150 ㎖ 전송 가방에 휴대 제품을 주입. </li></ol> 2. 준비 및 사용 자동화의 세포 농축 시스템 (재료 및 장비 표 참조) <ol><li> 셀 농축 시스템 (그림 1B)에 전환하고 프로그램 &quot;CCS_IFN-γ 심화&quot;를 선택합니다. 절차를 통해 운전자를 안내 지침과 함께 스크린 이미지를 나타내는 사용자 인터페이스를 관찰한다. </li><li> 매개 변수 &quot;운영자&quot;와 &quot;튜브 세트 P / N 번호&quot;를 입력합니다. 다음으로, 대화 형 모니터 화면에 표시되는 지시 사항에 따라 튜브 세트 (500) 자동화 된 세포 농축에 장치를 설치합니다. </li><li> 장치에 중간 버퍼를 연결하는 화면에 표시 단계별 지침을 따른다. 기록 카탈로그 번호와 connectin 전에 시약의 로트 번호악기에 들어. </li><li> 튜브 세트의 최종 점검 한 후, 펩티드 칵테일 가방의 클램프를 엽니 다. 매체 가방을 열고 튜브 세트의 자동 프라이밍을 시작합니다. </li><li> 프라이밍 공정이 완료된 후, 멸균 튜브 용접기의 도움으로 저장 주머니 (200 mL) 중의 염화나트륨 완충액으로 HSA (2.5 %)을 보완. 멸균 튜브 용접기를 사용하여 &quot;응용 프로그램 가방&quot;로 시작하는 휴대 제품을 전송합니다. </li><li> 어댑터를 통해 각각의 튜브에 CCS (IFNγ) 시약을 연결합니다. 농축 과정을하기 전에 세포 물질의 일부​​를 수집하기 원하는 시간을 입력합니다. 검토 및 모든 데이터의 정확성을 확인 / 입력 파라미터. 프로세스를 시작합니다. </li><li> 자동화 된 세포 농축 과정을 시작하기 전에, 품질 관리 가방 (QCB는, 원래의 분수 (오라)는 PBS / EDTA 버퍼로 희석하여 100 ㎖ 실 내용에서 약 1.3 ml에 포함)를 제거합니다. , QCB 인감 무게, 4 ° C에서 저장. </li><li> enrichme 시작NT 과정. 프로세스의 끝에서, 표적 세포는 저류 백에서 용출 완충제 대략 부피로 용출 될 것이다. </li><li> 비 대상 휴대 가방 (NTCB, 부정적인 부분 = NEG)와 대상 휴대 가방 (TCB, 긍정적 인 부분 = POS)를 밀봉하고 각 가방의 무게. 가중치는 나중에 세포 수의 계산에 사용한다. </li><li> 즉시 농축 과정을 거친 후 유동 세포 계측법 분석을위한 분수 당 두 개의 분취 량을 수집하고, 4 ° C에서 샘플의 나머지를 저장합니다. 세포 수의 결정과 농축 성능 분석 (표 1)에 대한 다른 샘플 나누어지는 하나의 샘플 나누어지는을 사용합니다. </li><li> 셀 농축 장비에서 설정 한 튜브를 제거합니다. 장래의 사용을 위해 USB 드라이브에 로그 파일을 전송. 참고 : 모든 시약은 무균 상태에서 준비를해야합니다. 바이오 안전성 II 형 후드의 사용을 적극 권장합니다. 건강한에서 분리 된 정상 상태의 성분 채집 세포 제품 (비는-동원)를 사용하여CMV 혈청 검사 양성인 공여자가 CMV 항원 특이 T 세포를 풍부하게합니다. 만 FDA는 HSA를 사용해야 라이센스. 셀 제조 버퍼 감소 순도와 표적 세포의 수율 감소를 초래할 것이다 낮거나 높은 주변 온도로 +25 ° C로 19 ℃에서 보관한다. </li></ol> 3. 세포 수의 결정 <ol><li> 표 1과 같이. 세포 수에 대한 QCB, NTCB 및 TCB의 분취 량을 4 ℃에서 10 분 동안 어둠 속에서 각각 나누어지는 (역가 1시 11분)에 CD45-VoBlue를 추가하고 품어. </li><li> 원래 분획 및 음 분획 양 분획 450 ㎕의 새로 제조 된 적혈구 세포 용해 액에 1.5 ml의 신선하게 제조 된 적혈구 세포 용해 용액 (1X)를 첨가하고, RT에서 15 분 동안 모든 분획을 배양한다. </li><li> (100 μg의 / ㎖로 희석 한 100) 직전에 분석, 1 μg / ML의 최종 농도로 요오드화 프로피 듐을 추가한다. 세포 수 및 VI를 결정하기 위해 자동 세포 계수기를 사용하여능력. 유동 세포 계측법 분석을위한 세포 카운터 장치 권장 소프트웨어를 사용합니다. 원래의 음과 양의 분수에 대한 백혈구의 절대 수를 결정합니다. 주 : 세포 수 분석 취한 시료 1 ㎖ 당 가능한 백혈구 세포 수는 세포 분석기 권장 소프트웨어를 이용하여 결정된다. </li><li> 그림 2 (지역 5, 가능한 백혈구)와 같이 영역을 설정합니다. 세포 수를 결정하기 위해 다음과 같은 게이팅 전략을 사용한다. 원래의 분수에서 가능한 백혈구는 그림 2에 표시됩니다. </li><li> 다음과 같이 표시 영역 (그림 2, 1-6) 계층은 다음과 같습니다 1 : 타임 게이트 → 2 : 단일 세포 → 3 : CD45 + 세포 → 4 : 백혈구 (파편 제외) 5 → : 실행 가능한 백혈구 → 6 : 실행 가능한 림프구 </li><li> 음과 양의 분수에 대한 세포 수를 결정하기 위해 동일한 단계를 반복합니다. 전체 분획물의 세포 수를 계산샘플 분획 (표 2)의 전체 부피의 희석 인자를 고려하여. </li></ol> 분리 성능 4. 시험 <ol><li> 사전 냉장 PBS / EDTA 버퍼 / 0.5 % AB 혈청 QCB, NTCB 및 TCB 분획 세포의 분취 액을 씻으십시오. 4 ℃에서 5 분 동안 300 XG에 세포를 원심 분리기와 뜨는을 대기음. </li><li> 포함 된 100 μL 항체, 형광 염색 혼합물에 재현 탁 세포 : CD3-FITC, CD4-APC, CD8-APC-Vio770, CD14-를 PerCP, CD20-를 PerCP, CD45-VioBlue 및 안티 IFNγ-PE (역가 1:11)과 4 ℃에서 10 분 동안 어둠 속에서 부화. </li><li> 1 ml의 신선하게 제조 된 적혈구 세포 용해 용액 (1X)를 첨가하고, RT에서 15 분 동안 배양한다. 4 ℃에서 5 분 300 XG에 원심 분리기와 뜨는을 대기음. PBS / EDTA 버퍼 / 0.5 % AB 혈청의 적절한 부피로 세포를 재현 탁. </li><li> 100 diluti 1 μg의 / ㎖ 직전 분석 (1의 최종 농도로 요오드화 프로피 듐을 추가) 100 μg의 / ㎖의에. 샘플의 순도를 평가하기 위해 유세포 분석을 수행한다. </li><li> CCS 농축 처리 후의 긍정적 분획에 도시 된 CD3 + T 세포를 결정하기위한 실행 가능한 백혈구 게이팅 전략 CD3 + T 세포를 계산하는 다음 게이팅 전략을 사용한다. 다음과 같이 표시 영역 (도 3a 및도 3b, 1-6)는 계층 적으로 연결되어있다 : 1 : 타임 게이트 → 2 : 단일 세포 → 3 : CD45 + 세포 → 4 : 세포 (파편 제외) 5 → : 실행 가능한 백혈구 → 6 : 실행 가능한 CD3 + 세포 인구 </li><li> CD4 +, CD8 +, CD4 + IFN-γ +와 CD8 + IFN-γ + CCS 농축 공정 후 T 세포 (표 2)의 주파수를 결정합니다. </li><li> F 아래 도시 CD4 +, CD8 +, CD4 + IFN-γ + CD8 + 및 IFN-γ + T 세포의 빈도를 판별 게이팅 전략을 사용또는 CCS 처리 후 원본과 농축 (캡처) 긍정적 인 부분. 다음과 같이 표시된 영역은 계층 적으로 연결되어 이름이 지정됩니다 : 1 : 타임 게이트 → 2 : 단일 세포 → 3 : CD45 + 세포 → 4 : 세포 (파편 제외) 5 → : 실행 가능한 백혈구 → 6 : 실행 가능한 CD3 + 세포 → 7 : CD4 + 세포 → 7A : CD4 + IFN-γ + 세포 (상자) → 8 : CD8 + 세포 → 8A : CD8 + IFN-γ + 세포 (박스) </li></ol> 주 : 링크 계층의 제 6 표시 영역 (1-6)도 3과 동일하며, 2 영역 최후로도 4 (6-8a)에 나타낸다.

<ol>
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M., Remberger, M., Alvin, O., Karlsson, H., Ringden, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Costs+after+Allogeneic+Haematopoietic+Sct+Are+Associated+with+Major+Complications+and+Re-Transplantation.">Increased Costs after Allogeneic Haematopoietic Sct Are Associated with Major Complications and Re-Transplantation.</a> Biol Blood Marrow Transplant. 18 (2), S339-S339 (2012).</li><li>Leen, A. M., 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=Multicenter+study+of+banked+third-party+virus-specific+T+cells+to+treat+severe+viral+infections+after+hematopoietic+stem+cell+transplantation.">Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation.</a> Blood. 121 (26), 5113-5123 (2013).</li></ol>

MD 앤더슨 암 센터 박사 쿠퍼 모두 ZIOPHARM 종양학, Inc. 및 Intrexon 회사에 재무 적 이해 관계를 가지고있다. 2015년 5월 7일에서 박사 쿠퍼는 ZIOPHARM 종양학의 최고 경영자 (CEO)로 임명되었다. 쿠퍼 박사는 지금 MD 앤더슨에서 방문 과학자입니다. 쿠퍼 박사가 설립 그는 인공 뉴 클레아와 Sangamo 생명 과학으로 특허를 보유 InCellerate, 주식을 소유하고있다. 그는 Targazyme, 주식 회사 (이전에 미국의 줄기 세포, 주식 회사), GE 헬스 케어, FERRING 제약, 운명 치료학, 얀센 제약, 브리스톨 - 마이어스 스 퀴브 (BMS)와 참조합니다. 그는 Cellectis의 과학 자문위원회에 있습니다. 그는있는 Miltenyi Biotec은에서 사례금을 받는다.

T 세포 대체 요법은 B 세포 악성 종양을 치료하기 위해 4 실행 가능한 옵션으로 떠오르고있다. 그 치료 가능성은 복제 성 노화 2 부족 표적 항원 특이 T 세포의 수를 주입에 의존한다. 이것은 현재의 양호한 제조 방법을 준수하여 확장 T 세포로부터 항원 특이 T 세포의 순수 인구를 선별함으로써 달성 될 수있다. 두 정렬 절차는 널리 즉, 형광 - 활성화 된 세포 (FACS)을 정렬 사?...

조혈 줄기 세포 이식 (HSCT) 1 이식편 대 종양 효과를 향상시키고, 기회 감염 (2)에 내성을 제공하는 T 세포 대체 요법과 결합 될 수있다. 주입을위한 항원 특이 기증자 유래 T 세포의 생성은 역사적으로 숙련 된 인력 및 GMP를 준수합니다 전문 시설의 사용을 요구하고있다. 이러한 T 세포의 전달 기회 감염 3 해상도뿐만 아니라 하부 암 (4)의 처리 결과이다. 최근 연구자들은 증명 그 천 단지 몇 바이러스 특이 적 T 세포의 대체 전송 (~ 1 × 10 4-2.5 × 105 세포 / kg받는 체중) 성공적 동종 조혈 모세포 5-9 후 편의적 CMV 감염을 치료할 수있다. 관련된 숙련 제조 요건 GMP 시설 제한된 수 및 셀 생산과 관련된 높은 비용 그러나 restr을 갖는다T 세포 치료 (10)를 약속에 icted 환자 액세스 할 수 있습니다. 항원 특이 적 T 세포를 분리하는 한 가지 방법은 CD45 및 IFN-γ을 인식하는 이중 특이 시약을 사용 CCS에 기초한다. 도시 된 바와 같이,이 방법은 자동화 된 세포 농축 CCS 장치 (도 1B)를 이용한 임상 등급 CMV 특이 T 세포를 생성하는데 사용될 수있다. CMV 특정 T 세포는 CMV 혈청 검사 양성인 기증자로부터 백혈구 성분 채집 총 핵 세포 (TNC)와 CMV pp65 유래의 항원에서 중복 펩티드를 배양에 의해 생성됩니다. 인간 백혈구 항원 (HLA)의 맥락에서 디스플레이 이들 펩타이드, IFN-γ를 분비하는 TNC 내의 CMV pp65 유래 특이 T 세포를 활성화. 이러한 T 세포는 &quot;캡쳐&quot;및 자기 적으로 분리 될 수있다. 제 세대 셀 농축 장치의 운전 (도 1a)는 복수의 착수 GMP 조건 하에서 세포 배양에서 숙련 된 사람이 필요하고, 직원의 코디&quot;캡처 된&quot;생성물을 생성하는 데 필요한 TEPS. 절차는 일반적으로 연속 운전의 12 시간에 10를 요구, 따라서 직원은 가능성이 GMP 시설에서 2 교대에 걸쳐 작업을 할 필요가있다. 이러한 제약은 이제 (도 1b에 도시 된) 제 2 세대의 장치에 의해 구현되어 없어진다. 이 장치는 1 세대 디바이스와 유사한 자기 농축을 수행하고 있지만, unbreached CCS 접근법의 다른 측면을 자동화한다. 이로 인해 공정의 대부분의 직원에 의해 자동으로 달성 될 수 GMP 팀의 부담을 감소시킨다. 장치는 밀폐 시스템으로서 동작하기 때문에, 항원 특이 적 T 세포를 포착하고 측량기를 시작하기 전에 백혈구 성분 채집 분리 및 재료의 제조에 포함 된 단계를 제외한 벤치 탑에서 처리 될 수있다. 전체 계측 및이 제 2 세대 세포 농축 장치의 기능의 세부 사항은 펍 된11 졌으. 여기서는 자동 세포 농축 CCS 시스템을 이용하여 정상 성분 채집 생성물로부터 CMV pp65 유래 특이 T 세포를 풍부하게하는 단계를 설명한다. 일단 격리 이들 CMV 특이 T 세포는 즉시 환자에 주입 될 수있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>CliniMACS PBS/EDTA Buffer 3 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>700-29</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Tubing Set TS 500</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-182</td>
			<td></td>
		</tr>
		<tr>
			<td>5 L waste bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>110-004-067</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Cytokine Capture System (IFN-gamma)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>279-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin (Human) 25%&#160;</td>
			<td>Grifols</td>
			<td>58516-5216-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer/Spike Interconnector</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-701</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9 % NaCl Solution (1 L)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS GMP PepTivator HCMV pp65</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for injections</td>
			<td>Hospira, inc, Lake Forest, IL</td>
			<td>NDC-0409-4887-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLEX GV Filter Unit 0.22 &#956;m&#160;</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>TexMACS GMP Medium 2 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-306</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Bag, 150 mL (for cellular starting material)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoMACS Freezing Bag 50</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>200-074-400</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL Syringes, sterile</td>
			<td>BD, Laagstraat, Temse, Belgium</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>CMV sero positive apheresis product</td>
			<td>Key Biologics, LLC, Memphis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Materials</td>
			<td>Manufacturer</td>
			<td>Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>AB Serum, GemCell</td>
			<td>Gemini Bio-Products, West Sacramento, USA</td>
			<td>100-512</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-FITC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-080-401</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-APC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-033</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8-APC-Vio770</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-065</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-072</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-077</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-VioBlue</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-136</td>
			<td></td>
		</tr>
		<tr>
			<td>aIFN-&#947;-PE, human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-940</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-PE</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-091-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide Solution (100 &#181;g/mL)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Device&#160;</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">200-075-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Software V1.0.0.RC</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer 10</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">130-096-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Software 2.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415R&#160;</td>
			<td>Eppendorf AG</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer K2</td>
			<td>Nexelom Bioscience, Lawrence, MA</td>
			<td>LB-001-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tubing welder SCDIIB</td>
			<td>Terumo Medical Corp., Elkton, MA</td>
			<td>7811</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated cell enrichment of cytomegalovirus-specific t cells for clinical applications using the cytokine-capture system

병원균 특이 T 세포의 대체 전송이 방지 및 동종 조혈 줄기 세포 이식 후 발생하는 감염 (CMV)와 같은 사이토 메갈로 바이러스 기회 감염을 치료하는데 사용될 수있다. 타사 도너 포함한 동종 공여자로부터 바이러스 특이 T 세포를 선택적으로 목적하는 T 세포를 전파 항원 구동 자극 반복 라운드를 이용하는 현재 우수 제조 CGMP ()을 준수 생체 전파 될 수있다. 항원 특이 적 T 세포의 식별 및 분리는 감마 인터페론 (IFN-γ)를 분비하는 활성화 된 T 세포의 사이토 카인 캡처 시스템에 기초하여 수행 될 수있다. 제조 공정은 시간 소모적이며, 숙련 작업자를 필요로하지만, 면역 복원 돕는 사이토 카인 캡쳐 시스템 (CCS)의 광범위한 인간 응용이 제한되어왔다. 이제 이러한 CliniMACS 프로디지 같은 2 세대 셀 농축 장치의 개발자동화 덜 노동 집약적 인 시스템을 이용하여 바이러스 - 특이 T 세포를 생성 할 수 있도록 연구자. 이 장치는 자기 임상 등급의 제품을 생성하기 위해, 자기 활성화 세포 분류 기술을 이용하여 표지 된 세포로부터 세포를 분리한다 표지, 폐쇄 시스템으로 설계되며, 벤치 탑에 액세스하여 조작 할 수있다. 우리는이 새로운 자동화 된 세포 농축 장치의 동작은 CMV 혈청 양성 공여자로부터 얻은 정상 성분 채집 생성물로부터 수득 CMV pp65 유래 특이 T 세포를 제조하는 것이 입증. 이러한 고립 된 T 세포는 다음 직접 기관 및 연방 정부의 규제 감독하에 환자에 주입 할 수있다. 적혈구의 제거를 비롯한 모든 생물 처리 단계는, T 세포, 항원 특이 적 T 세포를, 정제, 분리 및 세정의 자극은 완전 자동화된다. 이와 같은 장치는 애플리케이션에 대한 인간 T 세포 GMP (전용 우수 제조 밖에 제조 할 수있는 가능성을 높이기) 대신 시설은 직원이 여러 제품을 생산하는 자동화 된 프로토콜을 감독 할 수 혈액 은행 시설에서 생산 될 수있다.

이 연구에서, 자동화 된 세포 농축 CCS 시스템 CMV pp65 유래 특이 T 세포의 자동화 제조에 사용 하였다. CMV 특이 T 세포는 세 성분 채집 셀 제품에서 농축 하였다. 정상 상태의 성분 채집 제품은 10 총 10 핵 세포 (TNC)를 CMV 혈청 검사 양성인 기증자에서 2 시간에 걸쳐 수확 생성되었습니다. 10 9 TNC는 다음의 CMV pp65 유래 유래 펩티드 (60 nmol의) 4 시간 및 T 세포는 자동화 된 세포 농축 장치의...

Watch this Scientific Journal Video about Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System at JoVE.com

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

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

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

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

automated-cell-enrichment-cytomegalovirus-specific-t-cells-for

Research

JoVE Journal

Immunology and Infection

12.3K 조회수.  U.T. MD Anderson Cancer Center. The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

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

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

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

에 셀 운명을 따라<em&gt; E. 대장균</em&gt; 파지 람다에 의해 감염 후

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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 ...

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

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

펩타이드 : 에피토프 특정 T 세포의 MHC Tetramer 기반 강화

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

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

T 세포 양적 높은 처리량 단일 세포 세포 독성 분석

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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 ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

사용 모니터링 수지상 세포 이동<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H 자기 공명 영상

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

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

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

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

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

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

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

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

마우스 나이브 CD4<sup&gt; +</sup&gt; T 세포의 분리 및<em&gt; 시험관</em&gt; T 세포 하위 집합으로 차별화

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.