Authors were supported by the German Cancer Aid (Max Eder Nachwuchsgruppe, Deutsche Krebshilfe; EU), the LOEWE Center for Cell and Gene Therapy Frankfurt (EU, ST) funded by the Hessian Ministry of Higher Education, Research and the Arts, Germany (III L 4- 518/17.004) and by the "Alfred- und Angelika Gutermuth-Stiftung", Frankfurt, Germany (EU). BJ was funded by a Mildred Scheel postdoctoral scholarship from the Dr. Mildred Scheel Foundation for Cancer Research. ST was founded by a GO-IN postdoctoral fellowship (PCOFUND-GA-2011-291776). The authors thank Becton Dickinson (BD) for providing the FACSCanto II and Canto10c Flow Cytometry Analyzers used in this study.

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The authors have nothing to disclose.

설명 된 방법은 건강한 기증자 또는 환자의 전혈 시료로부터 NK 세포의 기능을 연구하기 쉽고, 빠르고 신뢰성있는 방법이다. 이 방법은 피 직접 전혈로부터 NK 세포를 정제 할 수있는 큰 이점을 제공하는 시간 소모적 인 많은 다른 정제 방법 15 필수 밀도 구배 원심 분리. 또한, 그것은 소아 및 / 또는 면역 결핍 환자의 샘플에 대한 적절한 대체한다 &quot;클래식&quot;NK 세포 분리 / 농축 방법에 비해 더 작은 샘플 크기를 필요로한다. 이 프로토콜은 NK 세포의 기능의 생체 외에서의 기저 값을 취득 할 수 있지만, 다른 방법에 상보 이전 8 기재된 이러한 방법으로 되, 상기 추가 NK 배양 및 확장 할 수있다. 그럼에도 불구하고 몇 가지 중요한 단계를 고려해야한다. 상기 분석은 역외 세포 내 항체 염색에 기초하기 때문에, 우와 최적를 결정하는 중요먼저 각 항체 농도를 rking. 특히 세포 내 염색을 위해, 사용 된 항체의주의 깊은 평가가 좋습니다. 좋은 방법이 사용 된 항체의 희석 계열을 테스트하는 것이다 (예를 들면, 1 : 12.5 : 100 (1)) 또는 이오 노마 이신 PMA /없이 자극 세포 현탁액이다. 이어서 자극되지 않은 샘플 간의 자극 포지티브 이벤트의 비율을 산출하고 플롯. 높은 양의 배 변화율 (최고 신호 대 백그라운드 비율)을 항체 희석 추가 실험 16을 위해 사용되어야한다. 또한, isotype- 및 FMO 컨트롤 같은 컨트롤의 사용은 긍정적 인 세포 집단에서 제대로 게이트 위해 세포 내 염색 특히 기본적인 것을 입증 할 수있다. 그러나, 설명 방법의 몇 가지 중요한 제한이 있습니다. CD107a 표현은 탈과립 마커이며, 따라서 간접적으로 NK 세포의 세포 독성을 나타냅니다. NK 세포 독성ND 탈과립 용량은 상이 할 수있다. 분비 그랜 자임 B의 열화 암세포 결과에서 자식 작용 경로 규제 접속 및 NK 세포 상호 작용 (17) 할 때 세포 사멸을 감소시킨다. 따라서 추가 DCM (예, 카스파 제 절단 3) 표적 세포 (18) 내에서 세포사 사건을 모니터링하기 위해 염색 패널에 첨가 될 수있다. 또한, NK 세포의 세포 독성은 세포 내 염색 (19, 20)에 의해 정량화 될 수있는 그들의 과립 내 독성 단백질 (예를 들면, 퍼포, 그랜 자임 B)의 양에 의해 영향을 받는다. 또한, 프로토콜을 변경하는 다른 가능성이있다. 대신 절연 NK 세포들, 말초 혈액 단핵 세포 (PBMC)를 사용할 수있다. 하나는 PBMC 집단 내의 NK 세포의 절대 수는 다양한 도너 사이에도 서로 다른 시점에서 동일 공여체 유래의 시료 사이에서 상이 알고 있어야한다하더라도. NK 세포 degranulT 비율 : ATION는 E에 크게 의존한다. 모든 실험 내내 T 비율 : 다른 기증자 간 또는 다양한 시점에서 NK 세포의 기능을 비교하기 위해, PBMC 인구 내의 절대 NK 세포 수는 상수 E를 설정하는 데 사용되어야한다. NK 세포의 기능이 동일한 도너 내의 상이한 시점에서 관찰되는 경우, PBMC를 동결 될 수 있고, 분석 간 실험 변동 (11)을 감소시키기 위해 한 번에 모든 상이한 시점에 대해 수행 될 수있다. 최대 18 색이 가능한 패널로 염색 때문에, NK 세포의 기능 분석은 훨씬 더 구체적으로 확장 될 수있다. CD57, NKG2A 킬러 세포의 면역 글로불린 형 수용체 (KIRs) 등의 추가 표면 마커를 이용하여, 상기 NK 세포의 서브 세트가 식별 될 수있다. 적어도 하나의 자기 KIR 표현 교육 NK 세포는 NK 세포 교양보다 K562 세포와의 상호 작용에 강한 degranulate. 반대로,보다 분화 된 CD57 + CD56 + </CD56 + NK 세포 (21) - SUP&gt; NK 세포는 덜 차별화 된 CD57에 비해 IL-12 및 IL-15의 자극에 따라 적은 IFN-γ를 생산하고 있습니다. 또한, NK 세포의 기능의 다른 마커는 해결 될 수있다. LFA-1이 NK 세포 유착에 중요한 분자 및 NK 세포 활성화시 개방 형태로 표현된다. 이것은 소위 &quot;내부 아웃 신호는&quot;초기 NK 세포 활성화 마커 (22)이다. 마지막 테스트 NK 세포의 기능에 대한 자극도 변형 될 수있다. 더 사이토 카인이 추가되지 않은 경우 우리의 경험에서 갓 K562 세포의 상호 작용에 만 degranulate 제대로 NK 세포입니다. 상기 사이토 카인을 첨가하지 않은 갓 절연 PBMC를 사용하기 때문에 와키 세포의 영향이 문제를 회피. 또한, 사이토 카인 생산, 특히 IFN-γ 및 TNF-α가 IL-12 및 IL-18의 자극 (23)에 개시 될 수있다. 북한군 셀 하위 집합 내에서 IFN-γ 생산은 dependin 다를 수사용 된 자극 (사이토 카인 대 목표에 의한 자극) 24 g. 대신 표적 세포로서 K562 세포를 사용 또한 종양 환자로부터 유도 된 일차 종양 세포는 전 또는 면역 조절 요법 (예를 들어, 모노클로 날 항체의 치료 또는 면역 조절 성 약제 중에자가 종양 세포에 NK 세포의 기능을 테스트하기 위해 사용될 수있다 (IMiDs)). 지난 몇 년 동안, 면역 요법의 분야는 면역 검사 억제제 (예 ipilumumab, nivolumumab, pembrolizumab)의 임상 승인 급변 한 아니라 T 세포의 발현 유전자 재조합 키메라 항원 수용체 (CAR)을 사용하여 25 성공적인 실험 혈액 암 환자 (27)의 치료 (참조 (26)에 검토) CAR-NK 세포 등. 따라서 NK 세포 면역 요법의 초점에 점점 더 많이 받고있다. 항 CD20 항체는 자원 처리에 큰 성공을 거두었습니다지난 20 년간 (28) 내에서 악성 림프종의 NT를. NK 세포의 대부분은 인식 항체를 표지 한 표적 세포 (- ADCC 항체 의존성 세포 독성)을 죽 세포를 활성화 낮은 친 화성 Fc 수용체의 CD16를 발현. NK 세포의 기능을 유발하는 항체의 능력은 기술 된 프로토콜을 사용하여 29 시험 관내에서 시험 할 수있다. Terszowski 등. 다른 임상 적으로 승인 된 항 CD20 항체를 이용하여 B의 임파 세포주에 대해 분석 NK 세포 &quot;ADCC. ADCC는 리툭시 맵 치료시 유발하지만 (신규 glycoengineered 타입 II CD20 모노클로 날 항체) 31 32 obinutuzumab 사용하는 경우, 이러한 효과가 관찰되지 않았 음 KIR / HLA 상호 작용에 의해 영향을 받았다 (제 임상 항 CD20 항체 (30)를 승인). 또한, NK 세포의 기능은 IMID 레 날리도 33과 다른 티로신 kinas 같은 다른 신규 한 항암 약물의 영향을 받는다전자 억제제 (TKI) 34. 현재 NK 세포의 특정 체크 포인트 억제제는 임상 시험에서 시험한다. 예 안티 KIR 35, 36 또는 항 NKG2A 항체 (NCT02331875)뿐만 아니라 항 CD137 항체 등 활성화 작용제 (; NCT02110082 NCT01775631)의 사용이다. 중요한 것은, 대체 NK 세포 전달은 전도 유망한 임상 효과 (37)와 다른 악성 질환의 다양한 수행되었다. 목적은 가장 도너를 선택하면, 환자의 종양에 대해 NK 세포의 기능 잠재적 NK 세포 공여자의 혈액 샘플을 사용하여 시험 관내에서 시험 될 수있다. 결론적으로, 설명 된 프로토콜은 건강한 기증자 또는 환자의 다양한 NK 세포의 기능을 분석하기 위해 설계된다. 이러한 기능은 선택된 시간 지점에서 다양한 자극에 따라 다양한 NK 세포의 서브 세트를 모니터링 할 수있다.

선천성 면역 시스템 자연 살해의 일환으로 (NK) 세포는 바이러스에 감염된 또는이 악 형질 전환 된 세포에 대한 방어의 첫 번째 줄에 기여한다. 억제 및 활성화 수용체 시스템은 T 세포와 대조적으로 종래 항원 프라이밍없이 건강하고 형질 전환 된 세포를 구별하도록 할 수있다. 대상 셀의 만남에 NK 세포는 자신의 목표를 죽이는 면역 시냅스에 자신의 세포 독성 과립 (예를 들면, 퍼포, granzymes)의 내용을 놓습니다. 타겟 셀시 (MIP-1β 예, 대 식세포 염증 단백질 1β) 및 케모카인 :; 또한, NK 세포는 다른 사이토 카인의 종류 (TNF-α, 종양 괴사 인자 α IFN-γ 예, 인터페론 γ)를 생성 및 분비 상호 작용 또는 사이토 카인 자극 한. 이러한 세포 독성, 케모카인 및 사이토 카인 생성 충분한 NK 세포의 기능은 다양한 DIS의 운명에 중요한 영향을 미칠용이하게합니다. 그들은 감소 IFN-γ 생산과 NK 세포 수용체 2를 활성화 감소 식으로 구성된 진단에서 결함이 NK 세포 프로파일을 나타내는 경우 백혈병 환자는 재발 속도를 증가 보여줍니다. 표적 세포의 상호 작용에 따라 사이토 카인 생성을 포함하여 NK 세포의 수 및 기능의 조기 회복 조혈 모세포 이식 3을받은 환자에서 재발 감소 향상된 생존율과 연관된다. 또한, C 형 간염 바이러스에 감염된 환자에게 인터페론 요법의 개시시 주변 NK 세포의 탈과립 용량 비 반응자 (4)보다 조기에 대응 강하다. NK 세포 수는 (&gt; 80 / μL) 임파종 또는 다발성 골수종을 앓고있는 환자에서는자가 줄기 세포 이식 (autoSCT) 후 15 일이 향상된 무 진행하고 생존율 5 예측된다. 흑색 종 환자에서 T 세포의 발현 및 immunoglobulin- 뮤신 - 도메인 containing 분자-3 (TIM-3), NK 세포에 대한 면역 조절 단백질은 병기와 예후 (6)와 상관 관계. 과학자들은 지난 수십 년에 걸쳐 NK 세포의 기능을 관찰 하였다. 앞서 프라이밍없이 종양 세포에 NK 세포 세포 독성의 초기 분석은 51 크롬 방출 분석을 사용하여 7 해결 하였다. 최근에 과학자들은 확장 된 NK 세포 (8)의 세포 독성을 평가하기위한 비 방사능 방법을 개발 하였다. 사이토 카인 및 케모카인 생산은 흔히 효소 면역 분석법 (ELISA) 방법을 사용하여 평가 9,10되었다. 지난 수십 년 동안 이러한 방법은 유동 세포 계측법 기반 분석에 의해 보완되고있다. 종래의면 염색 프로토콜과 함께 수송 단백질 억제제 (예 brefeldin A 및 넨신) 세포 permeabilization 방법의 사용은 다른 특정 림프에서 케모카인 및 사이토 카인 생성을 연구 과학자 활성화ocyte 서브 세트 (예를 들어, T, B 또는 NK 세포) 11. 또한, 다른 유동 세포 계측법 기반 분석법은 T 및 NK 세포의 세포 독성을 모니터링하도록 개발되었다. 2004 년 알터는 외. 독성 입자 (12)의 탈과립을위한 마커로서 표적 세포의 발생에 NK 세포의 리소좀 - 관련 단백질 CD107a (램프 1)의 표면 발현을 설명했다. 상이한 형광 색소 및 멀티 채널 유동 세포 계측기 다양한 우리 일에서 유효하기 때문에, 동시에 다른 NK 세포 서브 세트의 다양한 NK 세포의 기능 (세포 독성, 사이토 카인 및 케모카인 생산)를 모니터링 할 수있게되었다. 이 생검 또는 백혈구 감소증을 앓고있는 환자의 혈액 샘플에서 예를 들면 샘플 크기를 제한하는 상황에서 특히 중요해진다. 글로벌 NK 세포의 기능을 테스트하기 위해, 상이한 유동 세포 계측법 기반 분석을 효율적으로 결합 될 수있다. 테오 등. 난방에서 자극 NK 세포lthy의 종양 세포주 K562와 도너 13 유세포 통해 NK 세포 탈과립, 인사이드 - 아웃 신호 및 케모카인 생산을 분석 하였다. 최근 NK 세포의 하위 집단은 표현형 autoSCT 동안 종양 환자 기능 계측법 기반 분석법 흐름으로 분석 하였다. 또한 NK 세포 및 degranulate autoSCT 11 후 초기 시점에서 종양 세포를 인식시 사이토킨 / 케모카인을 생성 할 수 있다고 입증되었다. 여기 프로토콜 탈과립 용량, 케모카인 및 사이토 카인 생성을 포함하여 종양 세포가 가능한 동시에 상이한 서브 세트에서 NK 세포의 기능을 모니터 할 수 유동 세포 계측법 기반 분석법을 이용하여 상호 작용시 NK 세포의 기능을 평가하기 위해 설명된다.

<table><tbody><tr><td>RPMI 1640 + glutamine</td><td>Invitrogen</td><td>6187-044</td><td></td></tr><tr><td>penicilin/streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>BD Falcon Round Bottom Tube</td><td>BD</td><td>352008</td><td></td></tr><tr><td>fetal calf serum</td><td>Invitrogen</td><td>10270-106</td><td>heat inactivated before use</td></tr><tr><td>T-flask</td><td>Greiner Bio-One</td><td>690195</td><td></td></tr><tr><td>K562 tumor cell line</td><td>DSMZ GmbH</td><td>ACC 10</td><td></td></tr><tr><td>ammonium-chloride-potassium (ACK) lysis buffer</td><td>made in house</td><td>/</td><td>components are listed in the text</td></tr><tr><td>Distilled water: Ampuwa Sp&amp;uuml;ll&amp;ouml;sung 1,000 ml Plastipur</td><td>Fresenius Kabi</td><td>1088813</td><td></td></tr><tr><td>Megafuge 40R Centrifuge</td><td>Heraeus</td><td>/</td><td></td></tr><tr><td>EDTA blood collector tubes</td><td>Sarstedt</td><td>386453</td><td>S-Monovette 7.5 ml, K&lt;sub&gt;3&lt;/sub&gt;EDTA</td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>Hematopoietic media (XVIVO)</td><td>Lonza Group Ltd</td><td>BE04-743Q</td><td></td></tr><tr><td>human serum</td><td>DRK Blutspendedienst, Frankfurt/M&amp;nbsp;</td><td>/</td><td>healthy donors with blood type AB; heat inactivated before use</td></tr><tr><td>Neubauer-improved counting chamber, bright line</td><td>Marienfeld superior/ LO-Laboroptik Ltd.</td><td>0640030/ 1110000</td><td></td></tr><tr><td>trypan blue solution (0.4%)</td><td>Invitrogen</td><td>15250-061</td><td></td></tr><tr><td>3% acetic acid with methylene blue</td><td>Stemcell Technologies&amp;nbsp;</td><td>07060</td><td></td></tr><tr><td>Corning 96 Well Clear V-Bottom TC-treated Microplates</td><td>Corning&amp;nbsp;</td><td>3894</td><td></td></tr><tr><td>Falcon 96 Well Round Bottom Not Treated Microplates</td><td>Corning&amp;nbsp;</td><td>351177</td><td></td></tr><tr><td>DPBS (Ca&lt;sup&gt;2+&lt;/sup&gt;- and Mg&lt;sup&gt;2+&lt;/sup&gt;-free)</td><td>Gibco Invitrogen</td><td>14190-169</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A2153-100G</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>08591-1ML-F</td><td></td></tr><tr><td>phorbol 12-myristate 13-acetate (PMA)&amp;nbsp;</td><td>Merck</td><td>524400-1MG</td><td></td></tr><tr><td>ionomycin&amp;nbsp;</td><td>PromoKine</td><td>PK-CA577-1566-5</td><td></td></tr><tr><td>interleukin 15 (IL-15)</td><td>PeproTech</td><td>200-15</td><td></td></tr><tr><td>Proleukin S (IL-2)&amp;nbsp;</td><td>Novartis Pharma</td><td>730523</td><td></td></tr><tr><td>Golgi Stop, Protein Transport Inhibitor (containing Monensin)</td><td>BD Biosciences</td><td>554724</td><td>This product can be used in combination or instead of Golgi Plug. The best combination for the wished experimental setting has to be tested.</td></tr><tr><td>Golgi Plug, Protein Transport Inhibitor (containing Brefeldin A)</td><td>BD Biosciences</td><td>555029</td><td></td></tr><tr><td>paraformaldehyde</td><td>AppliChem</td><td>UN2209</td><td></td></tr><tr><td>saponin</td><td>Sigma Aldrich</td><td>47036</td><td></td></tr><tr><td>flow cytometer: Canto10C</td><td>BD Biosciences</td><td>/</td><td></td></tr><tr><td>FlowJo</td><td>TreeStar Inc.</td><td>/</td><td></td></tr><tr><td>Graph Pad</td><td>Graph Pad Inc.</td><td>/</td><td></td></tr><tr><td>MACSxpress Separator</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-308</td><td></td></tr><tr><td>MACSxpress NK isolation kit</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-185</td><td></td></tr><tr><td>MACSxpress Erythrocyte Depletion Kit, human</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-196</td><td></td></tr><tr><td>MACSmix Tube Rotator&amp;nbsp;</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-090-753</td><td></td></tr><tr><td>anti-human CD3 APC</td><td>Biolegend</td><td>300412</td><td></td></tr><tr><td>anti-human CD3 V450</td><td>BD Biosciences</td><td>560366</td><td></td></tr><tr><td>anti-human CD14 PerCP</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-094-969</td><td></td></tr><tr><td>anti-human CD14 V450</td><td>BD Biosciences</td><td>560349</td><td></td></tr><tr><td>anti-human CD16 PE</td><td>Biolegend</td><td>302008</td><td></td></tr><tr><td>anti-human CD16 PerCP</td><td>Biolegend</td><td>302029</td><td></td></tr><tr><td>anti-human CD19 PE-Cy7</td><td>Biolegend</td><td>302216</td><td></td></tr><tr><td>anti-human CD19 V450</td><td>BD Biosciences</td><td>560353</td><td></td></tr><tr><td>anti-human CD45 BV510</td><td>BD Biosciences</td><td>563204</td><td></td></tr><tr><td>anti-human CD56 FITC</td><td>Biolegend</td><td>345811</td><td></td></tr><tr><td>anti-human CD107a PE</td><td>Biolegend</td><td>328608</td><td></td></tr><tr><td>anti-human IFN-&amp;gamma; AF-647</td><td>BD Biosciences</td><td>557729</td><td></td></tr><tr><td>anti-human MIP-1&amp;beta; APC-H7</td><td>BD Biosciences</td><td>561280</td><td></td></tr><tr><td>DAPI</td><td>Biolegend</td><td>422801</td><td></td></tr><tr><td>Zombie Violet Fixable Viability Kit</td><td>Biolegend</td><td>423113</td><td>fixable dead cell marker</td></tr></tbody></table>

flow cytometry-based assay for the monitoring of nk cell functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

<html> 전체 NK 세포군 세 가지 NK 세포 서브 세트의 탈과립, 사이토 카인 및 케모카인 생산을 분석하는 게이팅 전략은도 1에 도시되어있다. 하나의 건강한 기증자의 대표적인 결과는도 2에 도시되어있다. 어떤 자극없이 NK 세포는 모두 IFN-γ도 MIP-1β을 생산하고 그 표면에 CD107a (그림 2A)을 표현하지 않았다. 대조적으로, 종양 세포 및 사이토 카인 자극 NK 세포는 세포 내 IFN-γ와 MIP-1β의 상당한 양의 생산. 또한, 그들 중 20 %는 그 표면 (도 2c)에 CD107a 표현으로 표시된 종양 세포의 상호 작용에 degranulated. PMA 및 이오 노마 이신 자극은 제어 (도 2B)을 사용 하였다. 전자 1 &quot;SRC =&quot;/ 파일 / ftp_upload / 54615 / 54615fig1.jpg &quot;/&gt; 그림 1 :. 전략을 게이팅 후속 게이트 플롯됩니다. 첫째, 파편은 FSC-A / SSC-A 플롯에서 제외됩니다. 그런 다음 림프구가 식별되고 이중선은 SSC-A / SSC-H 및 FSC-A / FSC-H 두 개의 플롯을 사용하여 제외됩니다. 그 후, 모든 CD45 + 세포와 모든 죽은, CD3 +, CD14 + 및 CD19 + 세포를 제외한 포함하는 게이트는 CD45 / 덤프 채널을 음모에 의해 설정된다. 다음으로, NK 세포는 CD56 + 세포에 게이팅로 식별됩니다. CD56 / CD16와 플롯 메인 NK 세포의 부분 집합을 식별하기 위해 사용될 수있다. 마지막으로, 전체 NK 세포 인구 내에서 기능 마커는 CD107a 각각 IFN-γ와 MIP-1β, 대 CD56 음모를 꾸미고로 식별됩니다. 이 NK 세포 하위 집합의 모든 다른 종류도 수행 할 수 있습니다. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54615/54615fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림 2.도 1에 기재된 게이트 전략을 사용하는 한 건강 NK 세포 공여자 대표 결과 (탈과립 용) CD107a 등 NK 세포 기능 마커는 IFN-γ와 MIP-1β가 식별 될 수있다. 왼쪽 열 (A)는 자극의 부재에서 NK 세포를 이용한 결과를 나타낸다. 가운데 열 (B)는 양성 대조군 PMA / 이오 노마 이신의 존재의 결과를 보여준다. 우측 컬럼 (C)은 IL-2 (100에게 U가 / ml) 및 IL-15 (10 ng의 / ㎖). 종양 세포주 K562 자극 NK 세포 및 사이토 카인의 존재 하에서 결과 제시 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54615/54615fig2large.jpg" target="_blank">여기 링크를 클릭하면 보는을 이 그림의 더 큰 버전.</a> <img의 고도 = &quot;표 1&quot;SRC = &quot;/ 파일 / ftp_upload / 54615 / 54615table1.jpg&quot;/&gt; 표 1 : 순도 검사에 대한 항체. <img alt="표 2" src="/files/ftp_upload/54615/54615table2.jpg" /> 표 2 : 역외 및 세포 내 염색에 대한 항체.

Watch this Scientific Journal Video about Flow Cytometry-based Assay for the Monitoring of NK Cell Functions at JoVE.com

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

간단하고 신뢰할 수있는 방법은 상이한 NK 세포의 서브 세트 내의 탈과립, 사이토 카인 및 케모카인 생산 등의 NK 세포의 기능 세트를 분석하기 위해 여기에 설명된다.

흐름 NK 세포의 기능을 모니터링 분석을 기반 세포 계측법

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

flow-cytometry-based-assay-for-the-monitoring-of-nk-cell-functions

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

20.8K 조회수.  Johann Wolfgang Goethe-University.  간단하고 신뢰할 수있는 방법은 상이한 NK 세포의 서브 세트 내의 탈과립, 사이토 카인 및 케모카인 생산 등의 NK 세포의 기능 세트를 분석하기 위해 여기에 설명된다. 

비디오: Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

준비 및 사용 HIV - 1 차 감염된 CD4 + 대상 세포로 T 세포 자연 킬러 세포 세포 독성 Assays에서

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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면역학 및 감염병학

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

급성 골수성 백혈병의 대량 하위 집단으로부터 줄기 세포를 구별하기 위해 NKG2D 리간드 표면 검출의 두 개의 흐름 세포 측정 접근법

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

암 연구학

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

인간 NK 세포 활동의 평가 대 한 흐름 Cytometry 기반 세포 독성 분석 실험

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

랫 트 태 반 천연 킬러 세포 세포 용 분해 기능의 평가를 위한 플레이트 기반 세포 독성 분석

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

치료 항체의 존재에 FcγRIIIa 참여에 의해 구동 인간 자연 킬러 세포 이벤트의 평가

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

의학

Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

Source: McBride, J. E., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54779">Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood.</a> J. Vis. Exp. (2017).
This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.