Name: rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development
Uploaded: 2018-06-19T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

우리는 우리의 기술 도우미에 게 빚을: 미국 형제와 H. Shkarlet, 실험 절차 동안 우리를 도운. 이 작품은 EU 보조금 (UniVax, 계약 번호 601738, 및 TRANSVAC2, 계약 번호 730964), 및 헬름홀츠 협회 부여 (하이-IDR)에 의해 부분적으로 투자 되었다. 자금 출처 조사에 영향을 주지 않았다 디자인, 원고 또는 게시를 위해 제출 하는 결정의 세대.

이 연구에 사용 된 모든 마우스 C57BL/6 배경에서 했다. 모든 동물 병원 체 자유로운 조건에서 유지 했다. 모든 실험은 (TierSchG BGBl 독일 동물 보호 법률의 규범에 따르면 수행 했다. 내가 S 1105; 25.05.1998) 허가 번호 33.4-42502-04-13/1281 및 162280에서 색 소니의 낮은 위원회 동물 실험의 윤리 및 국가 사무실 (낮은 색 소니 국가 사무실의 소비자 보호 및 식품 안전)에 의해 승인 했다.
1. CFSE OT의 얼룩-나 T 세포와 입양 전송
참고: OT-난 마우스 transgenically 생성된 동물 고정된 α와 β 사슬 함께 OVA의 면역 지배 펩 티 드를 인식 하는 T 세포 수용 체 (TCR)를 표현 하는 SIINFEKL10,11. 결과적으로, 이러한 마우스 수가 상당히 높은 SIINFEKL 전용 CD8+ T 세포 (97%)12 13정상 또는 OVA-예방 접종을 마우스 (≤ 1%) 비해.
<ol><li>추적 CD8의 절연 OT에서+ T 세포-나 쥐 T 림프 구 특정은 Thy1는는 (Thy1.1) 대립 유전자 표현:<ol>
<li>6-9 주 된 OT를 안락사-나 CO2 흡입에 의해 쥐 뒤에 자 궁 경부 전위14. 피부 외과 플라이어 집게의 도움으로 몸에서 피부를 분리 수술가 위로 절단 하 여 비장 및 주요 림프절 (사 타 구니, 겨드랑이, 자 궁 경부 쌍만) 해 부 고에 넣어 접시 (60 x 15 m m) 각각 포함 하는 100 µ m 기 공 메쉬 컵 조직 연 삭 및 셀 릴리스에 대 한.</li>
		<li>얼음에 있는 완전 한 RPMI 매체 (RPMI 1640, 10 %v / v, FCS 100 U/mL 페니실린, 50 µ g/mL 스)의 3-5 ml 접시 (각각 하나의 메쉬 컵)를 유지 합니다.</li>
		<li>주사기 나 비슷한 무 균 악기의 도움으로 장기 매 시 (사전 절단 불필요) 15 mL 원심 분리기 튜브에 결과 단일 세포 현 탁 액을 수집.</li>
		<li>300 x g 4 ° c.에서 10 분 동안에 세포 현 탁 액을 원심 폐기는 상쾌한. 원심 분리에 의해 차가운 PBS의 10 mL에 세포를 세척 하 고 다시 염화 암모늄 버퍼 (ACK 버퍼, 상용)의 1 mL/비장에 펠 릿을 일시 중단 하 여 비장에서 적혈구를 lyse. 얼음에 1.5 분에 대 한 셀을 품 어 하 고 이후 원심 분리 (으로 이전)에 의해 차가운 PBS의 10 mL로 세포를 씻어.</li>
		<li>다시 1 ml의 PBS (pH 7.2) 포함 5% 태아 둔감 한 혈 청 자석 절연에 대 한 같은 튜브에 펠 릿을 일시 중단 합니다.</li>
	</ol>
</li>
	<li>마그네틱 격리를 수행 하려면 CD8의 부정적인 선택을 진행+ T 제조업체 지침에 따라 자기 절연 키트를 사용 하 여 셀 또는 프로토콜15게시.</li>
	<li>CFSE 얼룩을 수행 하려면 먼저 CD8 수 계산+ T 세포 OT에서 얻은-난 자동된 셀 카운터 (입자 카운터)를 사용 하 여 마우스. 1-5 107 셀/mL 15 ml 송 골 매 관에 빛에서 보호 하는 37 ° C에서 7 분 5 µ M PBS에 CFSE와 1-5 mL의 볼륨에 배 얼룩.</li>
	<li>CFSE는 세포를 소 태아 혈 청의 같은 볼륨 (1:1)를 추가 하 여 얼룩을 냉각 하 고 빛 으로부터 보호 하는 37 ° C에서 추가적인 7 분에 대 한 그들을 품 어. PBS의 10 mL와 함께 셀을 두 번 세척.</li>
	<li>자동된 셀 카운터를 사용 하 여 셀을 계산 합니다. 3-5 100 µ L에 106 셀/마우스 배를 정 맥 주사 3-5 107 셀/mL PBS의 배을 셀 수 설정 (i.v.) 꼬리 정 맥을 통해.</li>
	<li>꼬리 정 맥 주사를 수행 하려면 적절 한 restrainers에 마우스를 고정. 다시 영역 및 쥐 꼬리 정 맥 혈관 확장을 허용 하도록 1-3 분 거리 20 ~ 25 cm 사이 붉은 빛 램프를 사용 하 여 주입에 쥐의 꼬리를 따뜻한. 참고:이 쉽게 정 맥 검색 및 세포 현 탁 액의 관리에 도움이 됩니다.</li>
	<li>(손으로 쥐와 램프 사이 배치) 확인 그것 너무 쥐에 대 한 뜨거운 고 꼬리 정 맥 명확 하 게 표시 될 때, 주입은. 셀 25 게이지 바늘161 mL 주사기를 사용 하 여 측면 또는 지 느 러 미 꼬리 정 맥에 주사.</li>
</ol>2. 예방접종 (보조 + OVA도 무료)
<ol><li>OT와 이식 생쥐를 배치-마 취 챔버 (1.7, 그림 1단계) 세포와 산소에서 isoflurane 마 취 기계14에 의해 관리.</li>
	<li>마우스 마 취에서 완전히 잠들 때, 약 실에서 그것을 꺼내와 gluteus superficialis (측면 허리)를 통해 깨끗 한 주사를 수행 하기 위해 전기 머리 트리밍 기계를 사용 하 여 영역에 마우스의 모피를 면도 와 응용 분야의 좋은 보기.</li>
	<li>백신, 25 게이지 바늘을 사용 하 여 면도 영역에 사우스 캐롤라이나의 50 µ L을 주입.</li>
</ol>3. 림프 톨 및 흐름 Cytometry 분석에 대 한 얼룩의 격리
<ol><li>림프 톨 및 splenocytes의 격리<ol>
<li>뒤에 자 궁 경부 전위 공동2 흡입에 의해 예방 접종된 마우스를 안락사.</li>
		<li>배출 및 먼 림프절과 비장 추출 하 고 조직 연 삭 및 셀 출시 100 µ m 기 공 메쉬 컵 포함 된 별도 접시에 그들을 배치. 1.1.3 통해 1.1.2 단계.</li>
		<li>원심 분리 후에 상쾌한 가만히 따르다와 다시 셀 펠 릿 잔해 PBS의 동일한 볼륨에 (약 100 µ L) 일시 중단.</li>
	</ol>
</li>
	<li>교류 cytometry 분석에 대 한 얼룩<ol>
<li>15 mL 원심 분리기 튜브에 표 1에 표시 된 농도에서 착 항 체를 포함 하는 마스터 믹스를 준비, 얼룩 믹스 집중 따라서 2 x 열매를 산출. 샘플 당 100 µ L를 마스터 믹스의 충분 한 볼륨을 준비 합니다. 셀 및 마스터 혼합 1:1 혼합 하 고 30 분 동안 4 ° C에서 품 어. 참고: 샘플 당 최종 착 볼륨 200 µ L (항 체 마스터 믹스의 셀 릿 + 100 µ L의 100 µ L) 이어야 한다.</li>
		<li>1.1.3, PBS의 10 mL를 두 번 추가에 설명 된 대로 셀 원심 분리에 의해 두 번 세척.</li>
		<li>다시 0.5-1에 얼룩진된 셀 중단 수집 및 전송 중단 교류 cytometer 튜브에 대 한 PBS의 mL. 항상 얼음 샘플을 유지 하 고 빛 으로부터 보호.</li>
	</ol>
</li>
</ol>4. Cytometry
<ol><li>항상 사전에 70-100 µ m 필터를 사용 하 여 셀 샘플 필터.</li>
	<li>표 1 (구슬 또는 셀)에 대 한 자세한 fluorophores에 대 한 하나의 얼룩 보상 컨트롤을 준비 합니다. 참고: 보상 한다 cytometer 또는 분석 소프트웨어17,,1819 에서 수행 되며 모든 샘플에 적용.</li>
	<li>그림2 제어 전략을 따릅니다. 앞으로 피해 지역 (그림 2A), 그리고 다시 사이드 분산형 및 측면 분산형 영역 (특수, 그림 2B) 남자를 제외 하려면 넓은 대 앞으로 살포 높이에 짧게, 게이트 인구.</li>
	<li>문 사실 CFSE 차별을 위해 BV 650 (자동-형광) FITC (CFSE) 대를 그려서 그림 2B 에서 인구 스테인드 높은 자동 형광 세포에서 세포. 구슬 또는 보상 컨트롤로 BV 650에 활용 된 항 체로 얼룩진 셀을 포함 합니다. 이 그림 (그림 2C) FITC 대 BV 650의 OT 게이트 하는 데 사용 됩니다-난 CFSE 셀 스테인드.</li>
	<li>받는 사람 마우스 대 기증자에서 파생 된 세포를 구별 하기 위해 APC (CD8) 대 Pe-Cy7 (Thy1.1-CD90.1-)에 대 한 그림 2 C 에서 인구를 게이트. 참고: 셀 OT에서 파생 된-난 기증자 마우스는 Thy1.1+ (CD90.1), 반면 WT (C57BL/6, 받는 사람) 마우스 파생 셀 Thy1.2+ (CD90.2) (그림 2D). 일부 연구소는 OT-난 Thy1.2 배경과 WT (C57BL/6)는 Thy1.1에 있는 쥐. 오티에 대 한 Thy1.2에 대 한 항 체를 사용 해야 하는 경우에-게이팅 셀.</li>
	<li>다시 CD8 게이트+ BV 450 (CD4)를 구성 하는 게이트에 APC (CD8) 대 그것을 그려서 Thy1.1+ 인구에서 세포 (그림 2E).</li>
	<li>인구는 CD8의 막대 그래프 디스플레이 사용 하 여 그림 2E 에 문이 표시+ (FITC, 530/15 채널-블루 레이저-), CFSE를 보여주는 셀 게이트 증식된 인구 10에서 농도 포함 하 여 수준2 곳 전적인된 제어 (농도 10 ~5, CFSE 얼룩이 지 고 교류 cytometer에서 전압 설정의 효능에 따라)는 (그림 2F).</li>
	<li>죽은 세포 확산 평가를 병행 평가 추가 게이트 (그림 2에에서는 표시 되지 않음) 플로팅 FITC 대 L/D 마커 (450/20 채널, UV 레이저)를 확인 합니다.</li>
	<li>취득 보상 컨트롤에 대 한 적어도 5000 이벤트 및 교류 cytometer에서 샘플에서 10.000 이벤트 (게이트 E, 그림 2). 낮은 또는 중간 흐름에는 cytometer 실행 (20.000 이벤트/s의 유량을 초과 하지 않는).</li>
</ol>

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Part B, Clinical cytometry. 72, 8-13 (2007).</li><li>Newrzela, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+receptor+diversity+prevents+T-cell+lymphoma+development.">T-cell receptor diversity prevents T-cell lymphoma development.</a> Leukemia. 26, 2499-2507 (2012).</li><li>Iwasaki, N., 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=Allergen+endotoxins+induce+T-cell-dependent+and+non-IgE-mediated+nasal+hypersensitivity+in+mice.">Allergen endotoxins induce T-cell-dependent and non-IgE-mediated nasal hypersensitivity in mice.</a> J Allergy Clin Immunol. 139, 258-268 (2017).</li><li>Tsuchiya, K., Siddiqui, S., Risse, P. A., Hirota, N., Martin, J. 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American journal of physiology.</a> Lung cellular and molecular physiology. , L54-L63 (2012).</li><li>Burgdorf, S., Scholz, C., Kautz, A., Tampe, R., Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+mechanistic+separation+of+cross-presentation+and+endogenous+antigen+presentation.">Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation.</a> Nature. 9, 558-566 (2008).</li><li>Last'ovicka, J., Budinsky, V., Spisek, R., Bartunkova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cellular immunology. , 79-85 (2009).</li><li>Oelke, 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=Functional+characterization+of+CD8(+)+antigen-specific+cytotoxic+T+lymphocytes+after+enrichment+based+on+cytokine+secretion:+comparison+with+the+MHC-tetramer+technology.">Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: comparison with the MHC-tetramer technology.</a> Scand J Immunol. 52, 544-549 (2000).</li><li>Wang, W., Golding, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytotoxic+T+lymphocyte+response+against+a+protein+antigen+does+not+decrease+the+antibody+response+to+that+antigen+although+antigen-pulsed+B+cells+can+be+targets.">The cytotoxic T lymphocyte response against a protein antigen does not decrease the antibody response to that antigen although antigen-pulsed B cells can be targets.</a> Immunology letters. 100, 195-201 (2005).</li><li>O'Sullivan, D., 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=Memory+CD8(+)+T+cells+use+cell-intrinsic+lipolysis+to+support+the+metabolic+programming+necessary+for+development.">Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development.</a> Immunity. 41, 75-88 (2014).</li><li>Xu, H. C., 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=Type+I+interferon+protects+antiviral+CD8++T+cells+from+NK+cell+cytotoxicity.">Type I interferon protects antiviral CD8+ T cells from NK cell cytotoxicity.</a> Immunity. 40, 949-960 (2014).</li><li>Volk, A., 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=Comparison+of+three+humanized+mouse+models+for+adoptive+T+cell+transfer.">Comparison of three humanized mouse models for adoptive T cell transfer.</a> The journal of gene medicine. 14, 540-548 (2012).</li><li>Safinia, N., 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=Humanized+Mice+as+Preclinical+Models+in+Transplantation.">Humanized Mice as Preclinical Models in Transplantation.</a> Methods Mol Biol. 1371, 177-196 (2016).</li><li>Grover, A., 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=Humanized+NOG+mice+as+a+model+for+tuberculosis+vaccine-induced+immunity:+a+comparative+analysis+with+the+mouse+and+guinea+pig+models+of+tuberculosis.">Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.</a> Immunology. 152, 150-162 (2017).</li></ol>

저자는 공개 없다.

현대 백신은 이상적으로 composedof 정화 항 리, 바이러스 같은 입자, 나노 입자 또는 라이브 벡터 처럼 전달 시스템의 가능한 추가 adjuvants. 백신을 디자인할 때 중요 한 부분은 임상 필요에 따라 올바른 보조 제를 선택 하는. 범위에 속하는 체액 세포 면역 응답 (또는 둘 다) 대, 조직의 면역 반응 (또는 둘 다), 대 로컬의 선거 및 백신 대상 모집단에 생성 해야 하는 메모리의 종류를 선호 포함할 수 있?...

세포 독성 T 세포 (CTL) 유도 하는 백신은 주요 치료 개입 싸움 암1의 특정 유형을 위해 개발 되었습니다. CTL은 또한 세포내 병원 체2에 대 한 예방 백신에 대 한 중요 합니다. 또한, CTL 누구 또한 초기 생활 감염5에 대처 하기 위해 CTL에 따라 신생아3,4 등 위험 인구에 기능적으로 활성 몇 면역 방어 메커니즘 중 하나입니다. 이와 관련, 백신 호흡 Syncytial 바이러스 (RSV)에 대 한 CTL 응답 (졸업생)을 유도 하지 않는 한 보조 제와 함께 개발 된 유아6에 감염 시 심각한 합병증에 지도 하는 백신의 완전 한 실패 귀착되는. 예방 접종의 이러한 부정적인 효과 CD8에 의해 반전 될 수 있다+ T 세포 응답7. 우리는 이전 인터페론 유전자 (스 팅) 주 작동 근의 어떤 자극에 의해 elicited (유형 I interferons) 주요 cytokines의 확산을 측정 하 여 일부 이러한 adjuvants8에 의해 생성 된 CTL 응답 필수적입니다 증명 하고있다 오티-예방 접종 및 기능을 유도 하는 CTL의 관찰에서 이러한 결과 사용 하 여 확장 예방 접종 일정9후 나 T 세포. OT의 확산의 측정-난 CD8+ T 세포는 야생 타입 (WT) C57BL/6 받는 사람 마우스 carboxyfluorescein succinimidyl 에스테 르 (CFSE) 염료 희석은 생성 하는 백신의 보조 능력의 강력한 추정 크로스-SIINFEKL, (ovalbumin, OVA의 면역 지배 펩타이드)의 프라이 밍 이 기술은 OT의 확산의 평가 위해 널리 이용 된다-내가 CD8+ 및 OT II CD4+ T 세포. 예를 들어 그것은 WT 동물에서 항 원 리콜 후 백신 효능을 측정 하거나 선택한 cytokines (코 쥐)의 부재에 사용 되었습니다. CFSE 스테인드 OT의 수동 전송 후 짧은 프로토콜 (실험 4 일)는 고안-난 CD8+ T 세포, 50의 1 개의 복용량의 구성 된 피하 (사우스 캐롤라이나) 면역 테스트 adjuvants 보충도 없는 OVA의 µ g 관리 (그림 1)입니다. 예방 접종 후 48 h 결과의 후속 CTL 응답을 생성 하는 보조의 용량의 신뢰할 수 있는 증거를 제공 한다. 이 전략에 의해 비장 (또는 먼 림프절) CTL 활동을 측정 하 여 응답의 범위 뿐만 아니라 예방 접종 후 배출 림프 노드의 로컬 면역 반응의 힘을 평가 가능 하다.

<table><tbody><tr><td>BD LSR Fortessa Cell Analyzer</td><td>BD</td><td>Special Order</td><td>Flow Cytometer</td></tr><tr><td>CFSE</td><td>Molecular Probes</td><td>C34554</td><td>Proliferation Dye</td></tr><tr><td>MojoSort Mouse CD8 T Cell Isolation Kit</td><td>Biolegend</td><td>480007</td><td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>Molecular Probes</td><td>L23105</td><td>Dead Cell Marker</td></tr><tr><td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td><td>eBioscience</td><td>25-0900-82</td><td>antibody</td></tr><tr><td>APC anti-mouse CD8a Antibody</td><td>BioLegend</td><td>100712</td><td>antibody</td></tr><tr><td>BV421 Rat Anti-Mouse CD4</td><td>BD</td><td>740007</td><td>antibody</td></tr><tr><td>Z2 coulter Particle count and Size Analyzer</td><td>Beckman Coulter</td><td>9914591DA</td><td>Cell counter. Z2 Automated particle/cell counter</td></tr><tr><td>EndoGrade Ovalbumin (10 mg)</td><td>Hyglos(Germany)</td><td>321000</td><td>Ovalbumin endotoxin free tested.</td></tr><tr><td>Cell Strainer 100&amp;micro;m nylon</td><td>Corning</td><td>352360</td><td>Cell strainer (100 &amp;micro;m pore mesh cups).</td></tr><tr><td>Sample Vials</td><td>Beckman Coulter</td><td>899366014</td><td>Sample vials for Z2 automated counter</td></tr><tr><td>C57BL/6 mice (CD90.2)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Company is now Envigo</td></tr><tr><td>OT-I (C57BL/6 background, CD90.1)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Inbreed at our animal facility. Company from where adquired is now Envigo</td></tr><tr><td>FACS tubes</td><td>Fischer (Corning)</td><td>14-959-5</td><td>Corning Falcon Round-Bottom Polystyrene Tubes</td></tr><tr><td>Falcon 15 mL tubes</td><td>Fischer (Corning)</td><td>05-527-90</td><td>Falcon 15mL Conical Centrifuge Tubes</td></tr><tr><td>PBS (500 mL)</td><td>Fischer (Gibco)</td><td>20-012-027</td><td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td></tr><tr><td>Red lamp (heating lamp)</td><td>Dirk Rossmann GmbH (Germany)</td><td>405096</td><td>Heating infrred lamp (100 wats)</td></tr><tr><td>IsoFlo (Isoflurane)</td><td>Abbott Laboratories (USA)</td><td>5260.04-05.</td><td>Isoflurane anesthesic (250 mL flask).</td></tr><tr><td>&lt;strong&gt;Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber&lt;/strong&gt;</td><td>Parkland Scientific</td><td>V3000PK</td><td>Isoflurane anesthesia machine.</td></tr><tr><td>RPMI 1640 medium</td><td>Gibco (distributed by ThermoFischer)</td><td>11-875-093</td><td>Base medium with Glutamine (500 mL)</td></tr><tr><td>Pen-Strept antibiotic solution (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>15-140-148</td><td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td></tr><tr><td>Fetal Bobine Serum (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>10082147</td><td>Fetal Bovine Serum, certified, heat inactivated, US origin</td></tr><tr><td>ACK Lysing Buffer (100 ml)</td><td>Gibco (distributed by ThermoFischer)</td><td>A1049201</td><td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td></tr><tr><td>Plastic Petri Dishes</td><td>Nunc (distributed by ThermoFischer)</td><td>150340</td><td>60 x 15mm Plastic Petri Dish, Non-treated</td></tr><tr><td>Cell Clump Filter</td><td>CellTrics (Sysmex)</td><td>04-004-2317</td><td>CellTrics&amp;reg; 50 &amp;mu;m, sterile</td></tr></tbody></table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

현대 소 단위 백신의 평가의 중화 항 체 생성은 중요 하지만 보조 선택에 대 한 충분 한 보여준다. 따라서, 세포 독성 T 세포 (CTL) 응답을 홍보할 수 있는 체액과 세포 면역 물질로 기능 adjuvants 절실히 필요 합니다. 따라서, 크로스 못쓰게 유도 하 고 이후 CTL 세대 강화 보조 후보자의 충실 한 모니터링 백신 개발에 중요 한 단계를 나타냅니다. 여기에 우리는 SIINFEKL 전용을 사용 하는 방법에 대 한 응용 프로그램을 제시 (OT-나)는 크로스-프레 젠 테이 션 모델 항 ovalbumin (OVA)는 비보에 다른 보조 후보자의 존재를 모니터링 하는 T 세포. 이 메서드는 adjuvants 최고의 크로스 못쓰게 기능을 선택 하는 빠른 테스트를 나타냅니다. CD8 확산+ T 세포 간 못쓰게의 가장 중요 한 표시 이며 그것은 또한 보조 유도 크로스-프레 젠 테이 션의 상관으로 간주 된다. 이 기능은 림프절과 비장 같은 다른 면역 기관에 평가할 수 있습니다. CTL의 정도 모니터링할 수 있습니다, 따라서 로컬의 특성에 통찰력을 주는 (주로 림프절 배수) 또는 조직의 반응 (먼 파선 또는 비장). 이 기술은 더 또한 종래와 유전자 변형 쥐의 다른 긴장에 사용 될 가능성을 제공 하 고 특정 크로스-프레 젠 테이 션 경로 억제할 수 있는 약물 테스트에 대 한 여러 수정 수 있습니다. 요약 하자면, 우리는 여기에 제시 하는 응용 프로그램 개발 하거나 백신 연구와 개발에 대 한 화학 adjuvants 수정 업계 또는 학계에서 백신 연구소에 대 한 유용할 것 이다.

Adjuvants (ADJ1 및 ADJ2)의 다른 조합을 사용 하 여 치료를 테스트 하려면 우리 adoptively 전송된 OT의 확산을 측정 하 여 CTL 세대 용량 평가-내가 CD8 cytometry (그림 2)에 의해+ T 세포. 이 위해, 우리는 이전 배출 림프절과 비장 (표 1)에서 격리 된 셀 스테인드. CD8의 확산을 측정 하 여 림프절과 비장,+ T 세포는 CTL 세대 고용량 ADJ2의 ?...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

선물이 여기 표준 면역학 기술에 대 한 응용 프로그램 (CFSE 스테인드 OT-내가 확산) 빠르게 보조 중재 된 세포 독성 T 림프 구 (CTL) 세대를 모니터링 하기 위한 vivo에서. 이 빠른 CTL 용량 추정 세포내 병원 체에 대 한 예방 백신으로 치료 암 백신의 개발에 대 한 유용합니다.

급속 한 비보에 평가 보조의 세포 독성 T 림프 톨 생성 기능의 백신 개발에 대 한

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

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Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K 조회수.  Helmholtz Centre for Infection Research. 선물이 여기 표준 면역학 기술에 대 한 응용 프로그램 (CFSE 스테인드 OT-내가 확산) 빠르게 보조 중재 된 세포 독성 T 림프 구 (CTL) 세대를 모니터링 하기 위한 vivo에서. 이 빠른 CTL 용량 추정 세포내 병원 체에 대 한 예방 백신으로 치료 암 백신의 개발에 대 한 유용합니다.

비디오: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

시험 관내 분석에서 HIV 특정 CD4 T 세포의 이펙터 기능에 면역 경로의 영향을 평가하기

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

연구

면역학 및 감염병학

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

T 세포 반응의 평가를위한 형광 대상 배열의 사용<em&gt; 생체 내</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

나노 입자 백신 접종 후 분석 항원 특이 CD8 + T 세포 반응에 대한 전체 동물 이미징 및 유세포 기술

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo에서 예방 접종 모델을 사용 하는 항 원 특정 T-셀 Cytolytic 함수의 검색에 대 한 분석 결과

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

Immunostimulatory 에이전트 평가: 림프 조직 추출 및 주입 경로-종속 수지상 세포 활성화

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

종양 특이 CD8+ T 세포 반응에서 면역 우세를 연구 하기 위한 생체 내 세포 독성 분석 법

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

비강 자기조립 나노에멀젼 종양백신의 제조, 특성, 독성 및 효능 평가 In Vitro 및 In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

체외 나노에멀젼 백신 보조제인 오피오포고닌 D의 세포 활성 평가

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

소 단핵구 유래 수지상 세포를 이용한 백신 면역원성 측정

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://app.jove.com/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

보조 매개 세포독성 T 림프구 생성을 모니터링하기 위한 면역학적 기술

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

급속 한 비보에 평가 보조의 세포 독성 T 림프 톨 생성 기능의 백신 개발에 대 한

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