이 작품은 미시간 대학 종합 암 센터의 윌과 Jeanne Caldwell 부여 연구 기금에 의해 지원되었다 부분적으로 NIH 보조금 CA82529 및 길슨 Longenbaugh 재단에 의해.

1. 알데플루어 염색
<ol><li>세포 샘플 준비: 배양된 종양 세포 또는 갓 수확한 종양 샘플에서 종양 세포의 단일 세포 현탁액을 준비합니다. 세포를 계산하고 ALDEfluOR 분석 버퍼에서 1 x 106 세포/ml의 농도로 세포 현탁액을 조정합니다.</li>
	<li>다음과 같이 컨트롤 튜브로 4 개의 12mm x 75mm 폴리스티렌 테스트 튜브라벨 : #1: 알데플루오르 #2, #3: ALDEFLUOR 플러스 DEAB, 및 #4: 7AAD.</li>
	<li>이러한 제어 튜브의 경우 1ml 셀 서스펜션을 #1 #4 넣고 튜브 #2 2ml를 넣습니다. 튜브 #3 5 μl DEAB (ALDH 억제제)를 추가하고 얼음을 유지합니다. 그런 다음 10 μl 활성 ALDEfluOR 기판을 튜브 #2 넣고 혼합물 1ml를 튜브 #3 즉시 전달합니다.</li>
	<li>동시에, 알데플루오 분석 버퍼에서 1 x 106 세포/ml에서 나머지 세포(샘플 튜브)에 백만 세포당 2μl 활성 ALDEFLUOR 기판을 추가합니다.</li>
	<li>37°C 수조에서 30분 동안 4개의 제어 튜브와 샘플 튜브를 모두 배양합니다.</li>
	<li>인큐베이션에 따라, 제어 관 #4 5 μl 7AAD를 추가하고, 샘플 튜브에 1 μl 7AAD 1 x 106 세포를 추가합니다. 4 °C에서 10 분 동안 배양하십시오.</li>
	<li>원심 분리기는 250 x g에서 5 분 동안 모든 튜브를 제거하고 상류체를 제거합니다.</li>
	<li>알데플루오 분석 버퍼에서 세포를 다시 중단합니다.</li>
	<li>유동 세포측정 기반 정렬을 수행합니다.</li>
	<li>DEAB로 처리된 ALDEFLUOR-스테인드 세포를 사용하여 선별 게이트를 음의 대조군으로 설정하고 생존 가능성을 조절하기 위해 프로피듐 요오드(PI) 7AAD-스테인드 셀을 설정합니다. 이러한 컨트롤에 기초하여, ALDEFLUOR+/ALDH높은,D5 및 SCC7 세포를 구별하기 위해 게이트가 설치되었다. 이러한 게이트는 ALDEFLUOR+/ALDH높은 D5 및 SCC7 세포를 위해 위에서 준비된 모든 샘플 세포를 정렬하는 데 사용됩니다.</li>
</ol>2. 암 줄기 세포 용해 성 수지상 세포 (CSC-TPDC) 백신 의 준비
<ol><li>CO2 및 상립 대퇴골 및 경골 뼈를 사용하여 마우스 (syngeneic B6 및 C3H, 각각)를 안락사시합니다.</li>
	<li>실온에서 1 분 동안 75 % 에탄올에 뼈를 넣습니다. 그런 다음 HBSS를 사용하여 뼈를 씻으시면 됩니다.</li>
	<li>뼈를 잘라 내고 21 G 바늘과 10ml 주사기를 사용하여 세포를 접시에 밀어 넣습니다.</li>
	<li>주사기를 사용하여 세포를 흡인시키고 날려 단일 세포 현탁액을 만듭니다.</li>
	<li>원심분리 후 1,500rpm에서 5분 동안, 상체를 폐기하십시오. 그런 다음 RBC 용해 버퍼 5ml를 추가하여 37°C 수조에서 1분 동안 RBC를 용해합니다.</li>
	<li>셀 수를 카운트하고 10 ng/ml GM-CSF 및 10 ng/ml IL-4를 포함하는 완전한 매체(CM)에서 1백만 개의 세포/ml 농도로 세포 현탁액을 조절한다.</li>
	<li>세포를 배양하고 CM 플러스 IL-4 및 GM-CSF를 3 일 후에 보상합니다.</li>
	<li>5일째에는 미숙한 수지상 세포(DC)를 수확하고 4.2ml 용액 C+ 1ml OptiPrep 밀도 그라데이션 배지를 함유한 DC 절연 배지를 준비한다.</li>
	<li>5ml CM의 세포 펠릿을 중단합니다. 셀 서스펜션을 격리 배지에 천천히 넣습니다. 실온에서 2,000 rpm의 원심분리기.</li>
	<li>CM과 격리 매체 사이에 DC를 수집합니다. 셀 수를 카운트하고 10 ng/ml GM-CSF 및 10 ng/ml IL-4를 포함하는 배양 배지에서 1백만 개의 세포/ml 농도로 세포 현탁액을 조절한다.</li>
	<li>배양 배지에서 ALDH높거나 정렬되지 않은 D5 또는 SCC7 세포를 일시 중단하고 급속동결 해동 노출에 4회 인하여 종양 용액을 준비한 다음 5분 동안 ~100 x g에서 스핀을 하여 용액의 막 부분을 수집한다.</li>
	<li>CSC-TPDC를 준비하려면 자가 알DH고세포의 용액으로 DC를 펄스합니다. H-TPDC를 준비하려면 분류되지 않은 이질성 종양 세포용재로 DC를 펄스합니다. DC와 종양 세포 리스의 비율은 동일합니다.</li>
</ol>3. 예방 접종 및 효능 평가
<ol><li>2,500D5 CSC-TPDC 또는 D5 H-TPDC 종양 세포를 피하(s.c)로 각각 정상 B6 마우스를 예방접종한다. 5,000 SCC7 CSC-TPDC 또는 SCC7 H-TPDC 종양 세포를 각각 접종하여 정상 C3H 동물 s.c.</li>
	<li>예방 접종 후, 이질D5 종양 세포 i.v. CO20 일 후 CO를 사용하여 마우스를 안락사시키는 이질D5 종양 세포로 B6 마우스에 도전하십시오. 폐를 수확하고 폐 전이를 예추합니다.</li>
	<li>SCC7 모델에서, DC 백신의 반대편에 .c 분류되지 않은 SCC7 종양 세포로 C3H 마우스에 도전한다. 종양 크기를 모니터링합니다.</li>
	<li>실험이 끝나면 CO2를사용하여 B6 및 C3H 마우스를 안락사시한다. 첫 번째 백신 후 34 일째에, CO2로마우스를 안락사시키고 동시에 무균 시술로 각 그룹의 비장을 수집합니다.</li>
	<li>고동된 안티 CD3 플러스 hrIL-2 또는 LPS 플러스 안티 CD40 (FGK45) mAb 배분을 포함하는 CM에서 안티 CD28 mAbs와 비장 T 및 / 또는 B 세포를 활성화합니다.</li>
	<li>활성화 후, 슈퍼 나츠퍼를 수집하고 ELISA 분석에 사용합니다.</li>
	<li>ELISA 분석의 경우, 4°C O/N에서 IFNγ, GM-CSF 및 IgG용 항체를 가진 코트 플레이트.</li>
	<li>차단 버퍼를 적용한 후 샘플 및 표준을 추가하고 실온에서 배양합니다.</li>
	<li>플레이트를 세척하고 인큐베이션을 위해 HRP 검출 항체 및 TMB 기판을 추가합니다.</li>
	<li>반응을 중지한 후 30분 이내에 450nm에서 ELISA 플레이트 판독기의 흡광도를 측정합니다.</li>
</ol>

<ol>
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STEMCELL Technologies는 오픈 액세스 게시 수수료를 지원했습니다.

SCID 마우스와 같은 면역 손상된 호스트는 호스트 내의 적응성 면역의 부족으로 인해 CSC의 면역 학적 평가를 배제합니다. 이 연구에서는, 우리는 면역 무능한 호스트에 있는 CSCs의 면역genicity를 평가했습니다, 이는 환자 설정을 더 밀접하게 모방할 수 있었습니다. 농축 된 CSC는 면역 원소이며 선택되지 않은 종양 세포 용해 성 DC에 비해 백신으로 그들의 lysates가 DC에 로드될 때 보다 효과적인 종양 보호 면역을 유도할 수 있습니다.  기계적으로, 보호는 CSC 반응성 항체 및 T 세포8의 선택적 유도뿐만 아니라 IFNγ 및 GM-CSF와 같은 타입-1 사이토카인의 생산에 의해 부여되었다.
수지상 세포 기반 백신 및 입양 T 세포 전달을 포함한 현재 면역 요법의 대부분은 종양-분화 항원들을 표적으로 하기 위해 고안되었습니다. 이러한 분화된 항원들을 발현하지 않을 수 있는 CSCs는 그러므로 이러한 면역학적 표적을 피할 수 있다. 대조적으로, 암줄기세포를 구체적으로 표적으로 하기 위하여 설계된 CSC 백신은 암세포의 이 특별한 인구를 파괴하고, 따라서 종양 재발 및 전이를 방지해서 백신의 치료 효험을 향상시킬 수 있다.
배양된 종양 세포와 갓 수확한 종양 모두에서, 우리는 높은 알데히드 탈수소 효소 활성에 기초한 혈류 세포측정에 의하여 CSC 농축 인구를 확인했습니다. 이러한 ALDH고세포는 CSC-TPDC를 생성하는 CC-TPDC를 생성하는 항원 소스로 사용되는 흐름 분류에 의해 단화될 수있다. 이 결과는 배양된 종양 세포에서 또는 임상 신청을 위한 갓 수확한 종양에서 고립된 CS를 사용하는 잠재력을 밝혔습니다.
임상적으로 관련성이 있기 위해서는 치료 환경에서 백신을 검사해야 합니다. 이 실험은 지금 우리의 실험실에서 수행되고 있습니다.

암 줄기세포는 종래의 화학요법 및 방사선 요법에 상대적으로 저항력이있다 1,2. 한편, 세포의 이 인구는 전통적인 암 치료1-4후에 암의 재발 그리고 진행을 책임지는 세포일지도 모릅니다. 암 줄기 세포에 분화 한 종양 항원의 발현의 부족으로 인해, 암 줄기 세포는 주로 분화 된 종양 세포에 항원을 대상으로 설계 암에 대한 치료의 현재 면역 학적 개입을 탈출 할 수있다. 따라서, 암줄기세포를 구체적으로 표적으로 하고 파괴하는 새로운 전략의 개발은 현재암 치료의 치료 효능을 증가시키는 약속을 지킬 수 있다. 이를 위해, 우리는 2개의 동물종양 (흑색종 D5 및 편평세포 암 SCC7)에서 암 줄기 세포 (CSC)를 풍부하게 한 인구를 격리하고, CSC-TPDC 백신을 준비하기 위하여 항원 제시 세포 (수지상 세포, DC)를 펄스하는 항원 근원으로 이용했습니다. 그런 다음 합성 면역 능력 호스트, B6 마우스 및 C3H 마우스에서 CSC-TPDC 백신에 의해 유도된 항종양 면역을 각각 평가하였다. CSC-TPDC 유도 항종양 효능은 이전에 우리 그룹5,6에서사용되어 온 분류되지 않은 이질성 종양 세포(H-TPDC)로부터 용액으로 펄스된 기존의 DC 백신과 비교되었으며, 다른조사자들(7)은 전임상 연구와 임상 시험 모두에서 비교하였다.

<table border="0" cellpadding="0" cellspacing="0" width="1022">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">ALDHEFLOUR KIT</td>
			<td style="width:399px;">Stemcell Technologies</td>
			<td style="width:219px;">1700</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine IL-4</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">214-14</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine GM-CSF</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">315-03</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse IFN-&#611; ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555138</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse GM-CSF ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555167</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">OptiPrep Density Gradient Medium</td>
			<td style="width:399px;">Sigma Aldrich</td>
			<td style="width:219px;">D1556</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">BD OptEIA TMB Substrated Reagent Set</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555214</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;"></td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Equipment</td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">BD FACS Aria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>336834</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Kcjunior</td>
			<td style="width:399px;">Bio-Tek Instruments</td>
			<td style="width:219px;">176058</td>
		</tr>
	</tbody>
</table>

generation of a novel dendritic-cell vaccine using melanoma and squamous cancer stem cells

우리는 암 줄기 세포 (CSC)-농축 된 인구는 Murine 흑색종 D5 합성C5/6 마우스와 편평상피암 SCC7 합성을 알데플루OR/ALDH를 마커로 사용하여 C3H 마우스에 합성, 맥박 세포에 항원 용액의 소스로 세포 용해성을 테스트 (DCs). ALDH높은 CSC lysates와 펄스 된 DC는 모델 과 폐 전이 설정 및 s.c 모두에서 분류되지 않은 전체 종양 세포 리스의 lysates로 펄스 DC보다 훨씬 더 높은 보호 항종양 면역을 유도한다. 종양 성장 설정, 각각. 이 현상은 CSC 백신 유도 된 유머뿐만 아니라 세포 항 CSC 반응 때문이었다. 특히, CSC-DC 백신을 실시하는 호스트로부터 분리된 비장세포는 분류되지 않은 종양 세포 용해 펄스-DC 백신을 실시하는 숙주로부터 분리된 비장세포보다 훨씬 더 많은 양의 IFNγ 및 GM-CSF를 생산하였다. 이러한 결과는 보조 제 환경에서 임상 사용을 위한 자가 CSC 기반 치료 백신을 개발하는 노력을 지원합니다.

ALDEFLUOR/ALDH는 다중 악성 종양에서 줄기 세포를 분리하는 단일 마커로서 사용되어왔다 8-11. 우리는 알데플루어를 마커로 사용하여 2개의 종양 모형 D5 및 SCC7에 있는 암 줄기 세포 농축인구를 확인했습니다. 우리는 뮤린 흑색종 B16-D5및 편평상피 세포암 SCC7에 있는 ALDEfluOR+ 세포를 검출했습니다. 우리는 ALDEFLUOR+ 세포가 배양D5 및 SCC7 종양 세포주에서 각각 약 0.5% 및 5.2%를 기여한다는 것을 것을을 발견했습니다(그림 1). 확립된 종양에서 갓 수확한 종양 세포는 ALDEFLUOR+ 세포의 존재를 확인하기 위해 분석되었다. 도 1에도와 같이, 생체 내에서 ALDEfluOR+ 세포의 2.5% 및 4.2%가 각각 D5 및 SCC7 종양을 확립하였다. 이들 선별된 D5 및 SCC7 ALDEFLUOR+/ALDH높은 집단의 종양성 및 자체 갱신 능력은 각각8개의합성 면역능력 숙주에서 평가되었다.
우리는 동물 연구 와 임상 시험 에서 모두 MC (H-TPDC)를 펄스하기 위해 이질성 무분류 종양 세포 용액을 사용하였다5,6. CSC의 면역원성을 조사하기 위해, 우리는 CSC-TPDC(그림 2)를생성하기 위해 CSC의 용액으로 ALDH높은 CSC 및 펄스 DC를 분리하고 CSC-TPDC가 종양 성장을 방지하는 데 유익한 효과가 있는지 테스트하기 위해 종래의 암 백신 제어로 H-TPDC를 사용했습니다.
우리는 CSC-TPDC에 의해 유도된 보호 항종양 면역을 검토해서 CSC의 면역원성을 평가했습니다. D5 모델에서, 순진한 면역 무능마우스는 CSC-TPDC 또는 H-TPDC (동일 lysate에서: DC 비율)를 가진 s.c 백신 접종을 했습니다. 제어 그룹은 식염수(PBS)를 받았습니다. 마지막 백신 후 1 주일, 마우스는 정맥으로 분류되지 않은 D5 종양 세포(즉,v.)로도전하고 폐는 폐 전이를 예매하기 위해 3 주 후에 수확되었다. 표 1에도시된 바와 같이, PBS 그룹과 비교하여, H-TPDC로 처리된 마우스는 더 적은 폐 전이를 개발하였다. 중요한 것은, CSC-TPDC로 처리된 마우스는 두 실험에서 H-TPDC 백신 단보다는 훨씬 적은 폐 전이가 있었습니다. SCC7 모델에서 정상적인 C3H 동물.c은 SCC7 CSC-TPDC 또는 H-TPDC를 각각 오른쪽 측면에 접종한 후, 분류되지 않은 SCC7 종양 세포s.c 좌측으로 도전하였다. PBS 그룹과 비교하여 H-TPDC는 종양 성장에 대한 겸손한 항종양 면역을 유도했습니다. 그러나, 대조군 및 H-TPDC 군(p<0.05, 도 3)과 비교했을 때 CSC-TPDC로 치료된 마우스에서 종양 성장의 현저한 억제가있었다. 이러한 결과는 CSC가 종양 세포의 도전에 대한 보호 면역을 유도하는 전통적인 분류되지 않은 종양 세포보다 DC를 로드하는 보다 효과적인 항원 소스로 사용될 수 있음을 나타냅니다.
관찰된 CSC 유도 된 보호 항종양 면역의 기본 메커니즘을 더 이해하기 위해 실험이 끝날 때 DC 예방 접종을 받는 동물로부터 비장을 수확했습니다. 비장 세포는 그 때 반대로 CD3/CD28/IL-2 또는 반대로 CD3/CD28/IL-2 + LPS/안티 CD40에 의해 활성화되었다. 이어서 배양 슈퍼나탈이 수집되어 사이토카인 및 항체의 발현을 검출했다.  D5 CSC-TPDC 또는 SCC7 CSC-TPDC(도 4)로 예방 접종된 동물로부터 비장 세포에 의해 IFNγ 및 GM-CSF의생산량이상당히 높았다. 더욱이, D5 CSC-TPDC 또는 SCC7 H-TPDC와 비교하여 D5 CSC-TPDC 또는 SCC7 CSC-TPDC로 예방 접종된 동물로부터 수집된 LPS/안티 CD40 활성화 비장 세포에 의한 IgG 생산량이 현저히(p<0.05) 높았다. 이들 항체는 각각 D5 및 SCC7 CSC에 결합하는 것으로 나타났으며, 이러한 결합은 보완8의존재속에서 CSC 리시스를 초래할 수 있다.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig1highres.jpg" src="/files/ftp_upload/50561/50561fig1.jpg"> 그림 1. ALDHFLUOR+/ALDH높은 인구는 새로 수확된 신선한 뮤린 D5 흑색종 및 SCC7 편평상피 세포 종양뿐만 아니라 배양에서 검출되었다. 50mmol/L DEAB로 처리된 종양 세포는 특정 ALDH 억제제로서, 음성 대조군으로 사용되었다.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> 
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig2highres.jpg" src="/files/ftp_upload/50561/50561fig2.jpg"> 그림 2. 수지상 세포 기반 암 줄기 세포 백신의 생성. CSC-TPDC 및 H-TPDC의 제조를 위해, 골수 유래 수지상 세포는 각각 ALDH높거나 분류되지 않은 종양 세포 용액으로 펄스되었다.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig2highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> 
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig3highres.jpg" src="/files/ftp_upload/50561/50561fig3.jpg"> 그림 3. CSC 용해 펄스 DC (CSC-TPDC) 백신은 s.c에서 보다 효과적인 보호 항종양 면역을 유도할 수 있었습니다. SCC7 종양 모델. <a href="https://www.jove.com/files/ftp_upload/50561/50561fig3highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> 

<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig4highres.jpg" src="/files/ftp_upload/50561/50561fig4.jpg"> 그림 4. CSC-TPDC로 예방 접종된 면역 능력 호스트에서 보다 강력한 전신 세포 반응.  비장세포는 H-TPDC 또는 CSC-TPDC 예방 접종을 받은 동물로부터 수확되었으며, 표시된 대로 활성화되었다. 배양 슈퍼넌트는 ELISA를 사용하여 사이토카인 검출을 위해 수집되었다.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig4highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells at JoVE.com

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

합성 면역 능력 호스트에서 평가, 암 줄기 세포 (CSC) 기반 수지상 세포 (DC) 백신은 이질 적인 대량 종양 세포와 펄스 전통적인 DC 백신 보다 상당히 높은 항 종양 면역을 입증.

흑색종과 편평상피암 줄기세포를 이용한 새로운 수지상 세포 백신의 생성

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

generation-novel-dendritic-cell-vaccine-using-melanoma-squamous

Research

JoVE Journal

Immunology and Infection

11.2K 조회수.  University of Michigan. 합성 면역 능력 호스트에서 평가, 암 줄기 세포 (CSC) 기반 수지상 세포 (DC) 백신은 이질 적인 대량 종양 세포와 펄스 전통적인 DC 백신 보다 상당히 높은 항 종양 면역을 입증.

비디오: Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

알레르기기도 염증 동안 폐 돌기 세포 성숙과 마이 그 레이션 분석

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

연구

면역학 및 감염병학

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipid 항원의 대량 Spectrometric 분석

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

전체 혈액 Phenotyping 분석을 사용하여 Multicenter 임상 시험을위한 주요 말초 혈 백혈구 인구의 열거

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

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

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

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

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

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

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

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

FDA 승인 베로 세포 백신 개발을위한 재조합 Arenavirus의 생성

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

정제 된 조혈 줄기 / 전구 세포에 DNA 돌연변이를 확인

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

호스트 세포 RN​​A의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

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

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

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

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 ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

골수성 창시자 및 정렬 전략 소설 셀을 사용 하 여 Murine 골에서 수지상 세포 선구자의 큰 숫자의 생성

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...