이 작품은 미국 소화기 학회 (AGA) 연구 학자 상과 크론과 미국 (CCFA) (ECS에) (SZS에) 경력 개발 상, NIH NIDDK의 F30의 DK089692의 대장염 재단과 위장 생물 노스 캐롤라이나 센터의 대학에 의해 지원되었다 과 질병 부여 P30의 DK34987 (조직학 코어). UNC 유동 세포 계측법 핵심 시설은 UNC Lineberger 종합 암 센터 NCI 센터 코어 지원 그랜트 (P30CA016086)에 의해 부분적으로 지원됩니다. 우리는 조직 병리학 적 분석과 면역 조직 화학 그의 도움을 동물 용 의약품의 노스 캐롤라이나 주립 대학에서 누가 B. BORST 감사합니다.

참고 : 모든 동물의 프로토콜에 의해 실험 동물의 관리 및 사용을위한 기관 동물 관리 및 사용위원회 (IACUC) 규정 및 국가 연구위원회의 가이드를 준수 승인되어 있는지 확인합니다. 기증자 마우스는 남성 또는 여성이 될 수 있지만,받는 사람 마우스는 남성이어야한다. 여성 수신자가 사용되는 경우, 도너 5 암컷 쥐이어야한다. 따라서, 마우스 5,6의 대장염 표현형을 이러한 장내 미생물에 영향을 미칠 수 있으므로, 정기적으로, 비 살균 침구 및 비 산성화 된 물을 사용하여 식민지를 유지합니다. 1. 실험 준비 <ol><li> 얼음처럼 차가운 미디어와 버퍼를 사용합니다. 실험을하는 동안 얼음에 세포를 보관하십시오. </li><li> 멸균 기술을 사용하여 멸균 생물 학적 후드에서 실험을 수행합니다. </li></ol> 비장 T 세포의 격리 2. <ol><li> 기증자 마우스 / CO에 마우스 자궁 dislocatio에 의해 2 챔버 따라 안락사N. 70 % 에탄올로 복부 스프레이. </li><li> 복부에 수평 절개를하고 복막을 노출 피부를 벗겨. 멀리 집게와 내부 장기에서 복막 잡고 노출과 비장을 절제하는 왼쪽 복부 복막에 절개를합니다. </li><li> 페트리 접시에 전체 미디어 10ml에 비장를 놓습니다. 제거하고 비장에서 초과 조직을 폐기합니다. </li><li> 분쇄 및 단일 세포 현탁액에 비장 떨어져 애타게 2 멸균 유리 슬라이드를 사용합니다. 필터 셀 50 ML 원뿔 튜브에 70 μm의 여과기를 통해 서스펜션과 전체 미디어의 5 ml로 여과기를 씻어. 한 50 ML 원뿔 튜브에 5 비장까지 놓습니다. </li><li> 7 분 450 XG에 원심 분리기 세포. 쏟아 또는 폐기물 용기에 진공 흡입에 의해 상층 액을 제거한다. </li><li> 조심스럽게 10 분 동안 실온에서 적혈구를 용해하는 용해 완충액 비장 당 5 ml의 세포를 재현 탁. 튜브 전체 미디어 동량 (비장 당 5 ㎖)를 첨가. </li><lI&gt; 원심 분리기 세포 7 분 450 XG에. 쏟아 또는 폐기물 용기에 진공 흡입에 의해 상층 액을 제거한다. </li><li> 부드럽게 라벨링 완충액 10ml에 세포를 재현 탁. </li><li> 트리 판 블루 배제하여 세포를 계산합니다. <ol><li> 세포 현탁액의 20 μl를 제거하고 미세 원심 튜브에 180 μL 트리 판 블루에 추가하고 잘 혼합. 5 분 후, 혈구와 현미경이 아닌 파란색 셀을 계산하는 세포를 표지 10 μl를 추가합니다. 가능한 세포의 수를 결정합니다. 세포 / 트리 판 블루 믹스를 폐기하십시오. </li></ol></li><li> 7 분 450 XG에 라벨링 버퍼 10 ㎖의 원심 분리기 세포. 쏟아 또는 폐기물 용기에 진공 흡입에 의해 상층 액을 제거한다. </li></ol> CD4 + T 세포의 보충 3. 참고 :이 섹션에서 사용되는 특정 제품에 대한 제조업체의 지침을 따르십시오. <ol><li> 부드럽게 감기 레이블 20 × 106 세포 / ㎖ 단일 세포 현탁액으로 세포를 재현 탁ING 버퍼. </li><li> 바이오틴 CD4 T 세포 농축 항체의 1 × 10 6 세포 당 5 μl를 추가합니다. 시료를 얼음에서 15 분 동안 세포를 배양한다. </li><li> 라벨링 버퍼의 10 배 볼륨을 추가합니다. 7 분 동안 450 × g에서 원심 분리기 세포. 조심스럽게 폐기물 용기에 진공 흡입을 사용하여 모든 뜨는을 대기음. </li><li> 철저하게 스트렙 타비 딘 - 복합 자성 입자를 소용돌이. 1 × 10 6 세포 당 입자의 5 μl를 추가합니다. </li><li> 철저하게 조심스럽게 섞는다. 30 분 동안 6-12 ℃에서 혼합 보관하십시오. </li><li> 20-80 × 106 세포 / ml의 농도로 라벨링 버퍼를 추가한다. 12 X 75mm 환저 시험관 당 세포 표지를 1.0ml까지 이동 ( &quot;포지티브 분획 튜브&quot;라고 함). </li><li> 6-8 분 동안 자석의 각 포지티브 분수 튜브를 놓습니다. </li><li> 자석에 여전히 긍정적 인 분율 튜브, 신중 (새로운 멸균 50 ML 원뿔 튜브에 유리 파스퇴르 피펫으로 포지티브 분수 관에서 referre을 뜨는 전송&quot;풍부한 부분&quot;) 등을 개발. 이 농축 분획은 CD4 + T 세포가 포함되어 있습니다. 자석에 매력을 표지 세포를 방해하지 않도록주의하십시오. </li><li> 재현 탁 된 세포를 피펫 팅 다운 격렬함으로써 단계 3.6과 같이 레이블링 완충액 동일한 체적 분률 포지티브 튜브에 남아. 6-8 분 동안 다시 자석에 긍정적 - 분수 튜브를 놓습니다. </li><li> 자석에 여전히 긍정적 인 분율 튜브,주의 깊게 자석에 부착 된 세포를 방해하지 않고 50 ㎖를 단계 3.8에서 원뿔 튜브를 멸균하기 위해 유리 파스퇴르 피펫으로 포지티브 분수 관에서 상층 액 (농축 분수, CD4 +)를 전송합니다. </li><li> 반복하여 얻은 CD4 + T 세포의 수율을 증가시키는 단계를 3.9-3.10. 농축 분획 (CD4 + 세포)를 사용하여 프로토콜을 계속합니다. </li></ol> 4. 라벨링 및 정렬 셀 (7) <ol><li> 원심 분리기는 7 분 동안 450 XG에 세포를 농축. 또는 진공 suct을 부어 상층 액을 제거한다폐기물 용기에 이온. </li><li> 레이블 버퍼 1 ㎖에 재현 탁 세포. 계산하는 단계 2.9에서와 같이 트리 판 블루 배제에 의해 세포 생존 능력에 대해 평가하기 위해 세포의 나누어지는을 제거합니다. </li><li> 10 × 106 세포 / ml로 라벨링 완충액 볼륨을 추가; 세포가 이미있는 경우 &lt;10 × 10 (6), 7 분 450 XG에 원심 분리기는, 폐기물 용기에 쏟아 또는 진공 흡입에 의해 상층 액을 버린다, 10 × 10 6 세포 / ㎖로 라벨링 버퍼의 볼륨을 추가합니다. <ol><li> 미세 원심 튜브에 이소 염색, 흠, 및 단일 염색 대조군 세포에 대해 약 5 ~ 10 × 10 5 세포 각각의 별도의 분취 량을 설정합니다. </li></ol></li><li> 세포에 5 ㎍ / ㎖의 CD4-FITC와 1 ㎍ / ㎖의 CD45RB-PE를 추가합니다. 미세 원심 튜브에 분주 적절한 동일한 농도로 이소 제어 얼룩과 하나의 얼룩을 추가합니다. 물론 조심스럽게 혼합하고 30 분 동안 빛으로부터 보호 얼음에 품어. </li><li> 세포 및 제어 라벨링 버퍼의 10 배 볼륨을 추가의. 7 분 동안 원심 분리기 450 XG. 폐기물 용기에 진공 흡입하여 상층 액을 버린다. </li><li> 4.5 단계에서 볼륨 레이블 버퍼에 재현 탁. 7 분 동안 원심 분리기 450 XG. 폐기물 용기에 진공 흡입하여 상층 액을 버린다. </li><li> 10 × 106 세포 / ml로 재현 탁 완충액 라벨링. FACS 튜브에 70 μm의 스트레이너를 통해 세포를 전달합니다. FACS를 위해 준비 될 때까지 빛으로부터 보호 얼음에 보관하십시오. </li><li> 설정하고 흠없는 세포와 단일 얼룩진 컨트롤 셀 소터에 대한 적절한 보상을 결정합니다. </li><li> 정방향 및 측면 산란 게이트 (그림 1A)와 생존 할 세포를 제외합니다. 이소 염색 컨트롤 CD4 +와 CD45RB + 세포 게이팅 설정합니다. CD4 + 인구에 게이트 셀. </li><li> 전체 미디어 2 ㎖와 튜브에 PE-염색 된 세포에 대한 간단한 히스토그램을 사용하여 높은 CD45RB 및 CD45RB 낮은 집단으로 분류 CD4 + 세포. 높은 CD45RB </su이 P&gt; 인구 + CD4 + 세포 CD45RB (높은 CD45RB)의 최고 40 %를 나타내고, 낮은 인구 CD45RB + CD4 + 세포의 CD45RB 최저 20 % (저 CD45RB도 1B). </li></ol><img alt="그림 1" src="/files/ftp_upload/52533/52533fig1.jpg" /> 그림 1 : FACS 분석 중 CD4 + CD45RB T 세포 인구의 플롯 세포 계측법 대표 흐름 (A - C) FITC CD4- 및 PE에 FACS으로 분류 된 기증자 C57BL / 6 마우스의 비장 세포를 CD45RB가 묻은 CD4 + CD45RB 높은 CD4 +. CD45RB 낮은 T 세포 인구. (A) 이중선 이벤트는 앞으로 산포도에서 제외 하였다. (B) 림프구는 전방 및 측면 산란 음모에 문이되었다. (C) CD4 + </SUP&gt; T 세포를 게이팅하고, (D) CD4 + CD45RB + T 세포 대 PE 이벤트 히스토그램 플롯 하였다. 낮은 CD4 + 세포 CD45RB CD45RB + 낮은 세포의 20 %로 간주 하였다. CD4 + 세포는 높은 CD45RB CD45RB + 세포의 최고 40 %로 정의 하였다. <ol start="11"><li> 인구의 순도를 평가하기 위해 FACS 시스템에서 각 셀 인구의 나누어지는을 실행합니다. </li><li> 원심 분리기는 7 분 동안 450 XG에 세포를 분류. 1 ml의 PBS에 재현 탁. 계산하는 단계 2.9에서와 같이 트리 판 블루 배제에 의해 세포 생존 능력에 대해 평가하기 위해 세포의 나누어지는을 제거합니다. </li></ol> 받는 사람으로 세포의 5. 주입 <ol><li> 재현 탁 4 × 106 세포 / ㎖ (CD45RB 높음) 또는 PBS에 2 × 106 세포 / ㎖ (낮은 CD45RB)에 셀을 정렬. </li><li> 새로운 멸균 튜브에받는 사람 당 CD45RB 높은 세포 100 μl를 전송합니다. PBS의 당 100 μl를 추가이 튜브에받는 사람. 따라서, 동물 당 총 주입량 200 μL, 그리고받는 사람 당 세포의 총량은 4 × 10 5 CD4 + CD45RB 고 나이브 T 세포이다. </li><li> T 조절 세포를 수신 한 실험군이 요구된다면, 새로운 멸균 튜브에 수신자 당 CD45RB 높은 세포 100 ㎕를 전송. 같은 튜브에받는 사람 당 CD45RB 낮은 세포의 100 μl를 추가합니다. 동물 당 총 주입 부피는 200 μL이다; CD45RB 높은 비율 : CD45RB 낮은 세포는 2 : 1입니다. </li><li> 고 / CD45RB 낮은 CD4 + 세포 복강 각받는 사람의 복부 (200 μL의 총)의 오른쪽과 왼쪽에 CD45RB 높거나 CD45RB 100 ㎕를 주입한다. </li></ol> 받는 사람 동물에서 질병 진행의 6. 모니터링 <ol><li> 받는 사람 동물의 임상 상태를 평가하고 다음 paramete 집계 임상 점수를 할당하려면주사의 날에 RS 8 주간 이후, 희생의 시간 : <ol><li> 체중 감량을 측정하여 낭비 결정 : 0 - 아니 체중 감소; 1 - 초기 체중의 0.1 ~ 10 %의 손실; 2 - 초기 체중 (도 2A)의 10 % 이상 손실. </li></ol></li></ol><img alt="그림 2" src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> 그림 2 : 염증의 임상 및 병리학 적 징후 Rag1에 야생 형 CD4 + CD45RB 높은 T 세포의 이동 이후에 발생하는 - / - 11 (A) NRDKO받는 사람 (5)에 의해 초기 체중의 평균 10 %에 손실 NRDKO받는 사람 마우스.이 주 전송 이후, Rag1 반면,- / -받는 사람은이 시간에 질병의 임상 증상을 나타내지 않았다. 각 점은 SEM ± 코호트에 대한 초기 체중의 평균 비율을 나타냅니다. **, P &lt;0.005. 직장 탈출증의 존재에 의해 입증되는 바와 같이 (B) 몇몇 마우스는 장내 심한 염증을 개발했다. 이 NRDKO받는 사람 마우스에서 직장 탈출증의 대표적인 사진입니다. (C) 육안에서 콜론 모두 Rag1 - / - 및 NRDKO받는 사람 마우스 Rag1에서 콜론에 비해 두꺼워 짧게 - / - 및 T 세포 입양 전송하지 않고 NRDKO 마우스. NRDKO받는 사람 마우스의 콜론은 심한 염증 증가 대장 무게 (데이터가 표시되지 않음)를 보여줍니다. <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <ol><li><ol start="2"><li> 이 사라질 때까지 깨끗한 용기에 동물을 배치하여 대변의 질을 결정의자 : 0 - 없음; 1 - 부드러운 의자; 2 - 물 및 / 또는 혈변. </li><li> 질병의 다음 증상의 존재에 의해 동물의 건강을 결정하지 : 0 - 더 웅크 리고 자세를, 모피, 또는 피부 병변을 곤두; 1 - 다음 현재의 어느 한 웅크 자세, 모피, 또는 피부 병변을 곤두. </li><li> 직장 탈출증의 존재 결정 : 0 - 결석을; 1 - 현재 (그림 2B). </li></ol></li><li> 그들의 초기 체중의 20 %를 손실 또는 제 도래 원하는 시점에서 CO 때 의해 경추 탈구이어서이 흡입을 희생이. 임상 질환은 주 5 사후 충만에서 시작 일반적으로 알 수있다. </li><li> 이전 5,7 설명 된대로 질병 심각도 평가합니다. <ol><li> 단계 6.1 8과 임상 점수를 할당합니다. </li><li> 대장의 길이와 무게 (그림 2C)를 측정한다. </li><li> 염증의 조직 학적 분석을 수행5. </li><li> 장 조직 절편 배양 9 자연 사이토 카인의 발현을 결정, 장간막 림프절은 10 노드, 및 / 또는 혈청 (11). <ol><li> 간단히, 이식편 문화 9, 길이 방향으로 개방, 희생 후 콜론을 제거하고 PBS와 깨끗한. 실온에서 30 분 동안 완전 배지에서 250 rpm으로 설정 한 궤도 진탕 기에서 인큐베이션 콜론. </li><li> 작은 조각으로 조직을 잘라 24 시간 동안 전체 미디어에 37 ° C에서 배양. 상층 액을 수집하고 ELISA 100 mg의 조직 당 사이토 카인의 정량을 위해 사용합니다. </li></ol></li><li> T 세포 표현형 및 / 또는 기능 (10)의 생체 특성을 수행합니다. </li></ol></li></ol>

<ol>
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The authors declare that they have no competing financial interests.

여기에서 우리는 면역 결핍 쥐에 CD4 + CD45RB + T 세포의 입양 전송에 의해 쥐에서 대장 염증을 유발하는 단계별 프로토콜을 설명합니다. 받는 사람 마우스 다른 균주 있지만 (예를 들어, BALB / C, 129S6 / SvEv, 비만이 아닌 당뇨병 (NOD)) 면역 결핍과 유전 적 모델 (예를 들어, SCID, Rag2 - / - 우리는 C57BL / 6 기증자 비장과 동계 Rag1을 사용 - / -)도 4,14-16 사...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

인간의 염증성 장 질환 (IBD), 자신의 장점과 단점 각각의 발병 기전을 연구에 사용할 수 많은 다른 동물 모델이 있습니다. 우리는 여기에서 T 및 B 세포 결핍 마우스에게 수신자 동계 비장 CD4 + T CD45RB 높은 세포의 대체 전송에 의해 개시되는 실험적 대장염 모델을 설명한다. 나이브 크게 이펙터 세포로 구성되어 CD4 + T CD45RB 높은 세포군 밀접 인간 IBD의 주요 측면을 닮은, 만성 장염을 유도 할 수있다. 이 방법은 질병의 발병과 진행의 측면을 연구하기 위해 조작 할 수 있습니다. 또한 그것은 장내 염증 즉, 타고난, 적응의 기능 및 규제 면역 세포 인구, 환경 노출의 역할, 미생물을 연구하는 데 사용할 수 있습니다. 이 문서에서 우리는 단계별 프로토콜 대장염을 유도하는 방법을 설명한다. 이 INCludes 성공적 연구 목적 대장염이 실험 쥐 모델을 개발하기 위해 필요한 키의 기술적 측면에 비디오 데모.

약 성체 C57BL / 6 마우스 공여체 비장에서 10 내지 10 × 106 CD4 + CD45RB 높은 T 세포를 확실하게 분리된다. 이 숫자는 나이와 변형 기증자 마우스 및 연구자의 능력에 따라 달라집니다. 때 4 × 6 CD4 + CD45RB 높은 T 세포는 C57BL / 6 Rag1로 전송됩니다 / 10 5 C57BL - / -받는 사람 마우스, 질병의 임상 증상은 주 5 사후 충만 주위에 등장 또는 마우스 (?...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

이펙터 CD4의 입양 전송하여 쥐과 장내 염증의 유도<sup&gt; +</sup&gt; CD45RB<sup&gt; 높은</sup&gt; 면역 결핍 마우스에게 T 세포

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

16.2K 조회수.  University of North Carolina at Chapel Hill. Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

비디오: Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

실험 전이 및 CTL 입양 전송 Immunotherapy 마우스 모델

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

연구

면역학 및 감염병학

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Murine Aortas 이내에 면역 세포의 흐름 Cytometry 분석

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Diabetogenic CD4의 입양 전송하여 마우스의 가속 제 1 형 당뇨병 유도 + T 세포

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

조혈 전구 세포의 입양 전송을 사용 Peyer의 패치에 쥐과 Plasmacytoid 수지상 세포의 개발을 평가

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

배려의 아교 모세포종 표준의 맥락에서 입양 치료에 대한 자동차 T 세포의 생성

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

분석<em&gt; 예 르시 니아의 enterocolitica</em숙주 세포 내로&gt; 이펙터 전좌 베타 - 락타 마제 이펙터의 용해 사용

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

마우스 비장에서 불변 자연 살해 T 세포를 분리하고 확장을위한 최적화 방법

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

마우스의 실험 세균성 폐렴의 유도 및 표현형에 대한 비 침습적 및 기술적 비 집약적 인 방법

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

마비 및 비주얼 시스템 부상 Neuromyelitis Optica Autoantigen Aquaporin-4에 대 한 T 세포 관련 하 여 쥐에서의 유도

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

정위 적 입양 전송 Orthotopic 인간 세포종 님 Xenografts Murine 모델에서 세포 독성 면역 세포의

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...