우리는 도움이 토론을 위해 아론 Selya 감사하고 싶습니다. 이 작품은 JS, MO 수있는 존스 홉킨스 말라리아 연구소에서 시험 부여 및 국방 부여 PC 040,972학과에 NIH 부여 AI29575, CA108835, AI077097 지원했습니다

1. 만들기, HLA - A2 - IG 기반 aAPC <ol><li> 무균 borate 버퍼 준비 <ol><li> 0.1M를 만들기 위해 물에 붕산을 디졸브. 7.0 산도를 조정합니다. </li><li> 4 0.22μm 멸균 필터와 매장을 통해 필터 ° C. </li></ol></li><li> 무균 비드 와시 버퍼 준비 <ol><li> 956ml PBS w / O 마그네슘 또는 칼슘을 가져가라. </li><li> 30ml 인간 AB 혈청을 추가합니다. </li><li> EDTA (에틸렌 다이아 민 테트라 초산) 2mM 최종 농도를 추가합니다. </li><li> 0.1gsodium azide를 추가합니다. </li><li> 4 0.22 μm의 살균 필터와 매장을 통해 필터 ° C. </li></ol></li><li> , 주식에서 약 400,000,000 구슬을 (비즈는 hemocytometer로 계산 가능) Invitrogen M - 450 에폭시 비즈의 1ml을 가지고, 살균, 스크류 최고 유리관에 넣어. </li><li> 비즈가 유리병의 측면을 준수하면서, Dynal 자석 MPC - 1에 대한 병을 넣어, 열망하여 뜨는을 제거하십시오. borate 버퍼의 1ml로 한 번 구슬을 씻으십시오. </li><li> 1ml borate 버퍼와 HLA - A2 - IG 이합체 및 안티 인간 CD28 mAb (클론 9.3) 20 μg 20 μg의 혼합물에 Resuspend 구슬. </li><li> 비드 활용하는 단백질 : 회전에 유리관을 넣고 4 회전 ° C를 24 시간 동안. </li><li> MPC - 1 자석에 튜브를 삽입하고 모든 borate 버퍼를 제거합니다. </li><li> 1ml 비드 와시 버퍼와 함께 두 번 구슬을 씻으십시오. </li><li> 1ml 비드 와시 버퍼에 비즈를 품어, 4 회전 ° C를 24 시간 동안. 비드 와시 버퍼 인간 AB 혈청가 포함되어 있기 때문에, 그것은 구슬의 모든 잔류 단백질 바인딩 사이트를 차단합니다. </li><li> 유리관에서 뜨는 제거 1ml 신선한 비드 와시 버퍼로 바꿉니다. </li></ol> 2. aAPC 및 펩티드 로딩, 저장 품질 관리 <ol><li> 외과 튜브의 버퍼를 세탁 100μl 외과에 ~ 5 × 10 5 구슬을 추가하고, 안티 - 마우스 IgG1 - PE의 1μl (HLA - A2 - IG의 FC 부분을 인식) 및 안티 마우스 IgG2a - FITC의 1μl (인식과 얼룩 안티 CD28 mAb의 FC 부분). 버퍼를 세척 외과에 20 분 얼룩 후, 흐름 cytometer (그림 4)에 의해 즉시 얼룩 결과를 다시 씻고 읽어보십시오. </li><li> 비즈에 펩타이드 로딩 : 1ml 멸균 PBS로 유리관에 두 구슬을 씻으십시오. 1ml 멸균 PBS로 Resuspend 다음 CMV 펩타이드 (1mg/ml)의 10μl 추가 </li><li> hemocytometer로 비즈를 카운트하고, 날짜와 농도로 병을 라벨. </li><li> aAPC는 HLA - A2 - IG 이합체에 펩티드 결합의 충분한 시간을 허용하는 4 최소 3 일 펩타이드 부화 ° C, 후 사용할 준비가되어 있습니다. 구슬이 4 저장할 수 있습니다 ° C와 적어도 6 달 동안 기능 남아있다. </li></ol> 3. 인간 CTL 절연 <ol><li> 10 BD 나트륨 헤파린의 Vacutainer 튜브로 건강한 HLA - A2 긍정적인 기증자 신선한 주변의 피 ~ 100ml를 수집합니다. 21 게이지 바늘 또는 용혈을 방지하기 위해 대형을 사용합니다. </li><li> 실온에서 10 분 300xg에서 원심 분리기 </li><li> 조심스럽게 흡인하여 최고 플라즈마 레이어를 제거합니다. 플라즈마는 문화 매체에 대한 보충으로 사용할 수 있습니다. </li><li> 멸균 PBS로 수집 플라즈마를 교체하고 살균 T75 문화 플라스크 또는 50ml 원뿔 튜브에 혈액을 전송합니다. 물론 최대 pipetting 아래로 PBS로 피를 섞는다. </li><li> 일단 모든 혈액 수집, 4 개의 추가 50ml 원뿔 튜브를 준비하고 Ficoll - Paque 플러스의 15ml를 추가합니다. </li><li> 천천히 오버레이 각 튜브의 Ficoll 위에 혈액 세포의 30 35ml. Ficoll과 혈액 세포 사이의 인터페이스는 별도. </li><li> 실온에서 20 분 500xg에서 원심 분리기. 브레이크 &quot;OFF&quot;와 레이어 사이에 명확한 인터페이스를 유지하기 위해 가능한 한 낮은 가속을 켭니다. </li><li> seriological 피펫을 사용하여 조심스럽게 PBMC 층을 기음과 신선한 50ml 원뿔 관에 PBMC를 수집합니다. 모든 PBMC 10 분 400xg에서 PBS와 스핀의 30ml 추가 수확하는 경우. 뜨는을 취소하고 모든 잔류 Ficoll을 제거 30ml PBS로 한 번 더 씻는다. </li><li> Miltenyi 인간의 CD8 + 제조 업체의 프로토콜에 따라 T 세포 격리 키트를 사용하여 CD8 + T 세포 격리로 진행합니다. 세포 -이 키트는 매우 (보통&gt; 95%) CD8을 파괴하여 CD8 + 세포에 대한 풍요롭게. </li><li> CD8 + T 세포를 계산합니다. 예상 순도는 95 %보다 커야한다. 이 사용 2x10 5 세포를 확인하고 CD4/3/8 외과 분석을 수행합니다. 나머지 CTL은 어느 항원 특정 aAPC 자극에 바로 사용할 수있는 또는 그들은 나중에 사용하기 위해 냉동 수 있습니다. </li></ol> 4. 체외 aAPC 기반의 문화 시스템에서 <ol><li> 문화 매체 준비 <ol><li> TF (국가 실험실 4 만들어진 T 세포 성장 인자) 2X 문화 매체 : 전체 RPMI 미디어 플러스 플라즈마 및 8% T 세포 성장 인자 autologous 5 % 기증자. </li><li&gt; 기증자 autologous 플라즈마 열 inactivated 인간 AB 혈청에 의해 대체될 수 있습니다. </li></ol></li><li> 전체 RPMI 매체의 TF 2X 문화 매체 플러스 8ml의 8ml에 Resuspend 1,000,000 CTL은 추가 1 × 10 6 aAPC은 잘 섞는다. </li><li> 물론 96 U 바닥 조직 배양 플레이트에 플레이트 세포에 멀티 채널 pipetter을 사용합니다. (사람마다 물론 160 μl) </li><li> 37 문화 세포 ° C, 7 일 동안 5 % CO 2 incubater. 80 μl / 잘 TF 2X 매체와 일 4 세포를 공급. </li><li> 세포는 하루에 7 수확 될 준비가 된 것입니다. 수확 후, 자석에 대한 세포를 배치하고 기존 aAPC를 제거합니다. </li><li> 항원 특이성은 제조 업체의 프로토콜에 따라 tetramer의 얼룩에 의해 결정하실 수 있습니다. 표현형의 얼룩 및 세포내 시토킨 얼룩은 우리의 이전 연구 3에 따라 수행됩니다. </li><li> 수확 세포는 동일한 조건에서 다시 aAPC와 replated 수 있습니다. 휴대폰 번호 및 항원 특이성은 반복 자극 후 증가 예정입니다. </li></ol> 5. 대표 결과 : HLA A2 - IG 및 안티 CD28 활용 이후 aAPC의 예제는 그림 4에 표시됩니다. 성공적인 단백질 결합에 해당하는 항체 얼룩의 명확한 변화가 분명하다. 주변 혈액에서 CMV 특정 CTL의 빈도가 aAPC - 중재 자극의 단일 주 후, 일반적으로 0.5-1 %이지만, 특이성은 55 도달할 수 - 93% (그림 2 및 3). 항원 특정 CTL의 확장은 다른 기증자 사이에 가변 수 있지만 결과가 동일한 기증자 내에서 재현할 수 있습니다. 추정함으로써, CMV 특정 세포의 확장 전구체 수준을 직접 예 생체내 (데이터가 표시되지 않음)에 비해 배 수천 수 있습니다. 세포내 시토킨 얼룩 (그림 3) 이러한 확장 CTL은 polyfunctional보다는 지친 있으며, 연장 세포 배양과 상당한 확산 후. 것을 보여줍니다 <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="그림 1"/> 그림 1. allogeneic HSCT의 입양 immunotherapy에 대한 인간의 CTL의 aAPC 기반 전직의 생체내 확장의 대표 플로우 차트 <img src="/files/ftp_upload/2801/2801fig2.jpg" alt="그림 2"/> 문화 일주일 뒤에 aAPC에 의해 생성된 특정 CMV CTL의 그림 2. 대표 tetramer의 염색법 결과 <img src="/files/ftp_upload/2801/2801fig3.jpg" alt="그림 3"/> 그림 3. aAPC에 의해 생성된 특정 CMV CTL 대표 세포 시토킨 얼룩 결과 (CMV의 특이성 61 %입니다) <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="그림 4"/> 그림 4. M - 450 에폭시 비즈 대표 얼룩 결과 반 마우스 IgG1 - PE 및 안티 마우스 IgG2 - FITC 물들일 단백질 활용 이후

<ol>
	<li>Walter, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N Engl J Med. 333, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리가 여기서 설명 aAPC 시스템은 항원의 다양한 인간에 대한 CTL의 전직 생체내 확장을위한 효율적인 시스템입니다. 특별한주의가 96 - 웰 플레이트 문화 단백질 결합의 품질과 aAPC과 CTL의 균등과 관련하여 이동해야합니다. 우리는 백만 배 4 항원 특정 CTL을 확장하는 동안, 이상 8 주 CTL을 확대할 수있다이 방법을 사용합니다. 셀 라인 또는 기타 acellular 플랫폼 5 활용한 다양한 인공 APC 시스템이되었습니다 있지만, 모든 시스템 확장 및 특이성에 대해서는 서로 다른 애플 리케이션을 지원과 함께 독특한 프로필을 가진 게시된 데이터에 따르면. CTL의 품질 수량만큼이나 중요되기 때문에 중요한 것은, 우리의 시스템에서 발생하는 CMV 특정 CTL의 polyfunctionality이 우수한 안티 바이러스 효율성을 부여 것으로 예상된다.

<table border="1"><tbody><tr><th> 시약 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> Vacutainer 튜브 (헤파린을 포함) </td><td> 백톤 디킨슨 </td><td> 367874 </td></tr><tr><td> 인간의 CD8 + T 세포 격리 키트 </td><td> Miltenyi </td><td> 130-094-156 </td></tr><tr><td> Dynabeads M - 450의 에폭시 </td><td> Invitrogen </td><td> 140.11 </td></tr><tr><td> Dynal MPC - 1 자석 </td><td> Invitrogen </td><td> 120 01D </td></tr><tr><td> Ficoll - Paque 플러스 </td><td> GE 헬스케어 </td><td> 17-1440-03 </td></tr><tr><td> RPMI 배지 1,640 </td><td> Gibco </td><td> 11,875 </td></tr><tr><td> HLA - A2 - IG 이합체 X </td><td> 백톤 디킨슨 </td><td> 551263 </td></tr><tr><td> iTAgMHC tetramer (HLA - A2 - CMV) PE </td><td> 베크맨 쿨터 </td><td> T20099 </td></tr><tr><td> 팰컨 분명 96 - 웰 플레이트 Microtest </td><td> 백톤 디킨슨 </td><td> 353077 </td></tr><tr><td> 쥐 반대 마우스 IgG2a - FITC </td><td> 백톤 디킨슨 </td><td> 553390 </td></tr><tr><td> 염소 방지 마우스 IgG1 - PE </td><td> Invitrogen </td><td> P21129 </td></tr><tr><td> 인간 혈청 타입 AB </td><td> 애틀랜타 생체 </td><td> S40110 </td></tr><tr><td> 마우스 안티 - 인간 CD8a - FITC </td><td> 시그마 - 알드리치 </td><td> F0772 </td></tr><tr><td> 마우스 안티 - 인간 CD8a - APC </td><td> 백톤 디킨슨 </td><td> 340684 </td></tr><tr><td> 마우스 안티 - 인간 IFNγ - FITC </td><td> 백톤 디킨슨 </td><td> 340449 </td></tr><tr><td> 마우스 안티 - 인간 TNFα - PE </td><td> 백톤 디킨슨 </td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

최적의 효과기 기능 CTL은 다양한 세포 내 감염 및 암에 대한 보호를 중재에 중요한 역할을 재생합니다. 그러나, 개인은 진압 면역 microenvironment을 전시 수 있으며, 활성화 CTL과는 달리 그들의 autologous 항원 제시 세포 tolerize하는 경향이 있거나 특정 항원 CTL을 anergize. 그 결과, 아직 실험 단계에 있지만, CTL 기반 입양 immunotherapy는 암 및 바이러스 감염 등 다양한 질환에 대한 유망한 치료되기 발전했습니다. 초기 실험에서 전 생체내 확대 CMV (사이토 메갈로 바이러스) 특정 CTL은 immunocompromised allogeneic 골수 이식 환자에서 CMV 감염의 치료에 사용되었습니다. 그것이 환자의 생명을 위협하는 CMV viremia의를하는 것이 일반적입니다받을 환자의 없음 CTL은 안티 CMV 내성이 adoptively 전송 CTL 1 설립 뜻, CMV와 관련된 질병을 개발 확대했습니다. 있지만 유망 결과는 흑색증에 대한 관찰되었으며 암이 다른 종류의 연장 수 있습니다. 인간의 CTL을 자극하고 확장 예 생체내 여러 가지가 있지만, 현재 방법은 비용과 기술적 한계에 의해 제한됩니다. 예를 들어, 현재의 금 본위 제도는 autologous DC의 사용에 따라 달라집니다. 이것은 각각의 환자가 leukocytes의 상당수를 기부 필요하며 비용도 아주 많이하고 힘드는입니다. 또한, DC 확장 CTL의 체외 특성화에 설명된이 단지 suboptimal 효과기 기능 3했다고 밝혔다. 우리가 입양 immunotherapy (그림 1) 인간 CMV CTL의 구체적인 예 생체내 확장을위한 고효율 aAPC 기반 시스템을 제시한다. aAPC 인간의 HLA - A2 - IG 이합체 및 안티 CD28mAb 4 셀 크기의 자석 구슬을 결합하여 만들어졌다. aAPC가 만들어진 후에는, 그들은 관심의 다양한 펩티드와 함께로드하고, 몇 달 동안 기능 유지하실 수 있습니다. 이 보고서에서는 aAPC는 CMV, pp65 (NLVPMVATV)에서 지배적인 펩타이드와 함께로드되었습니다. 일주일 동안 aAPC있는 건강한 기증자로부터 인간의 CD8 + CTL 투석을 culturing 후 CMV CTL은 특정 98% (그림 2) 특이성에 극적으로 증가하고 10,000 명이 넘는 배 증폭하실 수 있습니다. 더 CMV 특정 CTL이 필요한 경우, 추가 확장이 쉽게 aAPC과 반복적인 자극에 의​​해 얻을 수 있습니다. Phenotypic 및 기능 특성이 확장 세포 효과기 메모리 표현형을 가지고 있고 (그림 3) TNFα와 IFNγ 모두 상당한 금액을 보여줍니다.

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

유도 및 항원에 특정한 T 세포의 확장을위한 새로운 DC 독립적인 방법을 설명합니다. HLA A2 - IG 기반의 인공 항원 제시 세포 (aAPC)을 효율적으로 다양한 항원 특이성의 CTL 확장 HLA - A2 제한 펩티드와 함께로드됩니다. 이 기술은 CTL 기반 입양 immunotherapy에 대한 잠재력을 보유하고 있습니다.

효율에 대한 HLA - IG 기반 인공 항원 제시 세포 예 생체내</em인간 CTL의> 확장

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

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Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

15.3K 조회수.  Johns Hopkins University. 유도 및 항원에 특정한 T 세포의 확장을위한 새로운 DC 독립적인 방법을 설명합니다. HLA A2 - IG 기반의 인공 항원 제시 세포 (aAPC)을 효율적으로 다양한 항원 특이성의 CTL 확장 HLA - A2 제한 펩티드와 함께로드됩니다. 이 기술은 CTL 기반 입양 immunotherapy에 대한 잠재력을 보유하고 있습니다.

비디오: HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

항원 특정 VIVO에서</emCFSE 라벨 대상 세포를 사용하여> 죽이는 분석

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

연구

면역학 및 감염병학

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Bryostatin의 1/Ionomycin에 의해 종양 반응 T 세포의 생체내 전 확장 및 일반 감마 체인 크린 시토킨 제제

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

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 ...

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 ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

인공 항원은 세포 (aAPC) 중재의 활성화와 자연 킬러 T 세포의 확장을 제시

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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 ...

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 ...

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 ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

표현형 분석 및 대한 인간의 피부에서 림프구 분리<em&gt; 전의 VIVO</em&gt; 세포 배양

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

항원 중심의 성 대장염 모델의 개발은 항원이 T 세포에 세포를 제시하여 항원의 프리젠 테이션을 공부하기

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

효율에 대한 HLA - IG 기반 인공 항원 제시 세포 예 생체내 확장