이 작품은 ANR (ANR-11-LABX-0016-01)와 IHU Cesti 프로젝트 또한으로 자금을 관리 하는 "Investissements d'Avenir" 프랑스 정부 프로그램의 일부인 Labex IGO 프로젝트 (n ° ANR-11-LABX-0016-01)의 컨텍스트에서 실현 합니다 " Investissements d'Avenir"프랑스 정부 프로그램, 여는 프랑스 국가 연구 기관 (ANR) (ANR-10-IBHU-005) 관리. IHU-Cesti 프로젝트도 낭트 메트로 지구의 드 라 루아르에 의해 지원 됩니다.

저자 들은 아무 경쟁 금융 관심사 선언 합니다.

새로 이식할된 받는 사람으로 총 splenocytes의 입양 전송 규제 세포 유도 또는 치료에 의해 potentiated의 존재를 검색 하는 효율적인 방법입니다. 호스트 방사선-유도 된 과도 lymphopenia 전송 후 세포 생존 및 관용의 설립을 촉진합니다. 또한, 하위 치명적인 방사선 면역 재구성, 전염 성 관용34라는 현상 동안 새로운 규정 하는 세포로 변환 하는 tolerogenic 속성으로 셀에 대 한 시간을 떠?...

쥐의 심장 이식 관용 유도 및 유지 관리의 메커니즘을 해독 하는 관용 유도 치료를 평가 하기 위해 신뢰할 수 있는 장기 이식 모델 이며 기능적으로 유능 하 고 지배적인 규정 하는 세포를 유발 가능성이 있다. 프로토콜 아래 루이스 1A 받는 사람 쥐 (LEW.1A, RT1는는)으로 완벽 하 게 불일치 heterotopic 심장 이식 루이스 1W 기증자 쥐 (LEW.1W, RT1u)에서 설명합니다. 이 이식 조합에서 급성 거부 빠르게 (약 7 일)에서 발생 하 고 이식 박동 복 부의 촉진을 통해 측정 하 여 쉽게 평가 될 수 있다. 여기 우리는 쥐에 심장 이식에 관용을 유도 하는 세 가지 프로토콜을 제안 합니다. 이 모델에서 공차는 유도 및/또는 다른 규제 셀에 의해 유지. 첫째, CD40 CD40L 상호작용 CD40Ig 인코딩 하는 아 데 노 바이러스의 차단 CD8의 생성을 유도 하는 (AdCD40Ig)+ Tregs 허용 때 adoptively 유도 전송 보조 이식할된 받는 사람1. 또한, CD8의 고갈+ AdCD40Ig 치료 받는 사람 생성 된 Bregs 및 RegMCs2(와 반대로 CD8α 항 체) 세포. CD8의 깊은 분석+ Tregs 속성 인터 루 킨-34 (IL-34) 및 Fibroleukin-2 (FGL-2)3,,45,6로 정의 하는 여러 immunoregulatory 분자의 중요 한 역할을 강조 . 반면 (AAV 벡터)와 IL-34의 overexpression RegMCs의 세대를 통해 Tregs 유도, FGL-2의 overexpression 유도 Bregs, 규정 하는 세포의 복잡 한 네트워크를 기본.
만성 거부 천천히 개발 하 고 장기는, 때문에 만성 거부 대 공차를 구별 하는 심층 분석 필요 합니다. 이식 세포 침투에 대 한 평가 일반적으로 섬유 증, 혈관 벽 및 보완 C4d 증 착의 immunohistology7에 의해 두껍게. 조직학 방법 동물 희생을 요구 또는 생 검 이식, 여기 우리가 용납된 이식의 다양 한 기능을 평가 하는 간단한 방법 설명: 출현 그리고 규정 하는 세포와 혈액에서 항 기증자 특정 항 체 응답의 기능 cytometry에 의해 샘플 (여기, 우리 형광 활성화 된 세포 (FACS) 정렬 사용).
공차는 이식 치료의 체포 후의 유지 보수 규제 세포8의 유도와 일반적으로 연결 됩니다. 마지막 십 년간에서 연구에 초점을 맞춘 CD4+Tregs 만장일치로 그들 키 마커로 Foxp3+, CD25높은, 그리고 특징 CD127-9,,1011. 마찬가지로, 여러 마커 CD8에 기인 했다+ CD122 같은 Tregs+, CD28-, CD45RC낮은, PD1+, 헬 리오스+ 1,12,,1314 , 15 , 16 , 17. 년, GITR, CTLA4, 및 cytokines의 표현 (IL-10, TGFβ, 일리노이-34, IL-35, FGL-2)는 Treg 프로필3,4,6,13, 또한 연결 했다 18,19,,2021. 그러나, Bregs, RegMCs, 또는 NKTregs, 같은 신흥 규제 세포 인구 관련 특정 마커 부족합니다. 실제로, Bregs는 주로 보고 미 숙 CD24+ 세포, 모호한 CD27 식 및 때때로 IL-10, TGFβ, 또는 granzyme B22,,2324의 생산. 골수성 세포 계보의 복잡성 CD14, CD16, CD80, CD86, CD40, CD209a, 또는 CD16325,26등 규제 또는 proinflammatory 그들의 프로필을 정의 하는 여러 마커의 조합을 요구 한다. 마지막으로, 일부 마커 NKTregs CD11b 등을 식별 하기 위해 보고 된+, CD27+, TGFβ+, 하지만 더 많은 연구 하는 데 필요한 추가 phenotypically 설명27,28,29 ,30,,3132. 따라서, 진압 활동의 증거 추가, 새로운 바이오 마커의 식별 phenotypic 설명 합법화 하는 데 필요한 새로운 immunoregulatory 중재자, 새로운 세포 치료의 범위를 확장 하 고.
셀의 진압 활동을 평가 하는 두 가지 보완 방법 제안 한다. 첫째, 생체 외에서 메서드 표시 효과 기 T 세포 수용자 기증자 항 원 제시 세포 (Apc) 다른 비율 6 일 동안에 의해 자극된 된 진압 세포 배양으로 구성 되 고는 이펙터 분석 T 세포 확산을 기증자 감독 면역 억제를 반영 한다. 세포 치료 쥐에서 직접 셀에 순진한 쥐 및 진압 활동에 대 한 (또는 다른 규제 셀 인구), 치료 비 이식할된 쥐에서 억압: 이펙터 비율의 범위에서 비교할 수 있습니다. 또한,이 방법은 어떤 이식 필요 하지 않습니다 및 결과 6 일 이내에 얻을 수 있습니다. 둘째, vivo에서 메서드는 새로 비친된 이식할된 받는 사람에 게 의도 된 규정 하는 세포 치료 쥐에서 전송 이루어져 있다. B 세포, 골수성 세포 또는 T 세포에서 치료 비 순 쥐가 일반적으로 급성 거부 하 고 입양 전송 시 이식 생존을 머리말을 붙이는 수, 취급 받는 potentiated 진압 활동 세포는 이러한 특성 1,2,3,,433. Lymphopenia 받는 방사선에 의해 유도 된 혈액 항상성 영향을 받지 남아 adoptively 전송된 셀 안티 기증자 면역 응답을 보다 쉽게 습득 하도록 허용 하는 것이 좋습니다. 두 방법, 수용자의 생체 외에서 이용 가능 하 여 타사 Apc 또는 vivo에서 입양 양도 진압에 대 한 받는 사람 제 마음으로 투입으로 셀 안티 기증자 특이성의 분석을 수 있습니다. 하지만 비보에 방법 상당수 셀, 제대로 표현 해야 진압 활동 생체 외에서33셀 부분 모집단을 보다 쉽게 평가 수 있습니다.
체액 응답 또한 관용의 상태 및 기증자 항 원에 대 한 감독된 항 체 응답의 평가를 측정할 수 있습니다. 실제로, 공차 기증자 향해 체액 반응의 부재 하지만 새로운 항 원에 체액 응답을 개발 하는 받는 사람에 대 한 능력의 보존 및 메모리 응답의 보존에 의해 특징 수 있습니다. 첫째, alloantibody 검출의 원리의 기증자 세포 혈 청 부 화 이식할된 받는 사람에서 다음 받는 사람 항 체에 의해 기증자 세포의 인식에 기반. 둘째, 체액 응답 외 인 항 원에 감독의 장기 허용 받는와 열쇠 구멍 삿 Hemocyanin (KLH) 완전 한 Freund의 보조 제와 유화 평가 다음 자극 될 수 있습니다. 특정 항 원에 대하여 IgM와 IgG 항 체의 존재 감지할 수 있습니다 4와 13 일 각각, 면역, 효소 연결 된 ImmunoSorbent 분석 결과 (ELISA)34와 함께 다음. 셋째, 면역 메모리 응답의 보존-7 및 3 일 이식의 Rbc + 8 및 17 다음 이식 받는 사람 혈 청 일에 수집된으로 얼룩 xenogeneic 적혈구 (Rbc)의 주입에 의해 평가 될 수 있다. 이러한 모든 메서드는 특정 보조 항 체와 FACS 얼룩에 의해 미만 1.5 h에 결과의 신속한 취득 또는 ELISA에 의해 몇 시간을 사용 하 여 면역 글로불린의 식별에 대 한 수 있습니다.
마지막으로, 이러한 프로토콜 이식 모델의 특성을 위해 디자인 되 고 어느 정도까지, 자가 면역 질환 모델에 적용 될 수 있습니다. 방법의 원리는 모든 수 종 조 변경 될 수 있습니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LEW.1W and LEW.1A rats</td>
			<td>Janvier Labs, France</td>
			<td></td>
			<td>8 weeks old,&#160;</td>
		</tr>
		<tr>
			<td>BN third party donor rats</td>
			<td>Janvier Labs, France</td>
			<td></td>
			<td>8 weeks old,&#160;</td>
		</tr>
		<tr>
			<td>name</td>
			<td>company</td>
			<td>catalogue number</td>
			<td>comments</td>
		</tr>
		<tr>
			<td>reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AdCD40Ig</td>
			<td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td>
			<td></td>
			<td>home made plasmids</td>
		</tr>
		<tr>
			<td>IL34-AAV</td>
			<td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td>
			<td></td>
			<td>home made plasmids</td>
		</tr>
		<tr>
			<td>FGL2-AAV</td>
			<td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td>
			<td></td>
			<td>home made plasmids</td>
		</tr>
		<tr>
			<td>anti-TCR</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>R7/3 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD25</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX39 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD8</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX8 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD45RA</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX33 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD161</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>3.2.3 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD11b/c</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX42 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-TCRgd</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>V65 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD45RC</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX22 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD4</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX35 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD45R</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#554881, His24 clone</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgG-FITC</td>
			<td>Jackson ImmunoResearch Laboratories, INC, Baltimore, USA</td>
			<td>#112-096-071</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgG1</td>
			<td>Serotec</td>
			<td>#MCA 194</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgG2a</td>
			<td>Serotec</td>
			<td>#MCA 278</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgG2b</td>
			<td>Serotec</td>
			<td>#MCA 195</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgM-FITC</td>
			<td>Jackson ImmunoResearch Laboratories, INC, Baltimore, USA</td>
			<td>#115-095-164</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin HRP</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#554066</td>
			<td></td>
		</tr>
		<tr>
			<td>KLH</td>
			<td>Sigma Aldrich, St. Louis, USA</td>
			<td>#9013-72-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td></td>
			<td>Phosphate Buffer Solution without calcium and magnesium,&#160;</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma, Saint-Louis, USA</td>
			<td>#9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB substrate reagent kit</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#555214</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTraceTM CFSE cell proliferation kit</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium 1X</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#31870-025</td>
			<td></td>
		</tr>
		<tr>
			<td>penicilline streptomycine</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes Buffer</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>non essential amino acids</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#11360-039</td>
			<td></td>
		</tr>
		<tr>
			<td>2 beta mercaptoethanol</td>
			<td>Sigma, Saint-Louis, USA</td>
			<td>#M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Proliferation Dye eFluor&#174; 450 Cell</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#65-0842-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma, Saint-Louis, USA</td>
			<td>#G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche Diagnostics, Germany</td>
			<td>#11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma, Saint-Louis, USA</td>
			<td>#E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Fresenius Kabi</td>
			<td>#B230561</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic dynabeads</td>
			<td>Dynal, Invitrogen</td>
			<td>#11033</td>
			<td>Goat anti-mouse IgG</td>
		</tr>
		<tr>
			<td>One Comp eBeads</td>
			<td>Ebiosciences, San Diego, USA</td>
			<td>#01-1111-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Naplbuphine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Terramycine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Freund&#39;s adjuvant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer lactate</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis solution</td>
			<td></td>
			<td></td>
			<td>Dilute 8,29g NH4Cl (Sigma, Saint-Louis, USA A-9434), 1g KHCO3 (Prolabo 26 733.292) and 37.2mg EDTA (Sigma, Saint-Louis, USA E5134) in 800ml H2O. Adjust pH to 7.2-7.4 and complete to 1L with H2O.</td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td></td>
			<td></td>
			<td>Dilute 1g collagenase in 500 ml RPMI-1640 + 5 ml Hepes + 2% FCS</td>
		</tr>
		<tr>
			<td>PBS-FCS (2%)-EDTA (0.5%)</td>
			<td></td>
			<td></td>
			<td>Add 5 mL EDTA 0,1M (Sigma, Saint-Louis, USA E5134) and 20ml FCS to 1ml PBS 1X</td>
		</tr>
		<tr>
			<td>CFSE (Vybrant CFDA SE Cell Tracer Kit Invitrogen)</td>
			<td></td>
			<td></td>
			<td>Dilute 50&#181;g (=1 vial) of CFDA SE (component A) in 90&#956;l DMSO (component B) solution to obtain a 10mM stock solution. Then, dilute stock solution at 1/20 000 in PBS 1X to obtain a 0.5&#956;M solution</td>
		</tr>
		<tr>
			<td>complete medium for coculture</td>
			<td></td>
			<td></td>
			<td>500ml complete RPMI-1640 medium with 5 ml Penicillin (80 unit/ml)-Steptomycin (80 mg/ml), 5 ml L-Glutamine, 5 ml Non Essential Amino Acids (100X), 5ml Pyruvate Sodium (100mM), 5 ml HEPES buffer (1M), 2.5 ml b mercaptomethanol (7 ml of 2-bmercaptoethanol stock diluted in 10 ml RPMI), 10% FCS</td>
		</tr>
		<tr>
			<td>name</td>
			<td>company</td>
			<td>catalog number</td>
			<td>comments</td>
		</tr>
		<tr>
			<td>equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>falcon 50ml</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#227261</td>
			<td></td>
		</tr>
		<tr>
			<td>falcon 15ml</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#188271</td>
			<td></td>
		</tr>
		<tr>
			<td>sieve</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning plastic culture dishes</td>
			<td>VWR, Pessac</td>
			<td>#391-0439</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#181;m and 60&#181;m tissue filters</td>
			<td>Sefar NITEX, Heiden, Switzerland</td>
			<td>#03-100/44 and #03-60/35</td>
			<td></td>
		</tr>
		<tr>
			<td>96 wells U bottom plates for coculture</td>
			<td>&#160;Falcon U-bottom Tissue Culture plate, sterile, Corning</td>
			<td>#353077</td>
			<td></td>
		</tr>
		<tr>
			<td>96 wells V bottom plates for FACS staining</td>
			<td>ThermoScientifique, Danemark</td>
			<td>#249570</td>
			<td></td>
		</tr>
		<tr>
			<td>96 wells flat bottom ELISA plates</td>
			<td>Nunc Maxisorb</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>seringue for spleen crush</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#309649</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA reader</td>
			<td>SPARK 10M, Tecan, Switzerland</td>
			<td>SPARK 10M, Tecan, Switzerland</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bain marie&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X rays irradiator</td>
			<td>Lincolshire, England</td>
			<td>Faxitron CP160</td>
			<td></td>
		</tr>
		<tr>
			<td>solar agitator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Canto II</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Aria II</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>magnet</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>12302D</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro and in vivo assessment of t, b and myeloid cells suppressive activity and humoral responses from transplant recipients

이식에서 주요 관심사는 규정 하는 세포의 유도 통해 특정 공차를 달성 하기 위해입니다. 허용 오차 메커니즘의 이해 안정적인 모델을 필요합니다. 여기, 우리 costimulation 신호 봉쇄 또는 유전자 전송을 통해 immunoregulatory 분자의 upregulation에 의해 유도 된 쥐에 심장 이식에 관용의 모델을 설명 합니다. 이 모델의 각 규제 T 세포 (Tregs), 규정 하는 B 세포 (Bregs) 등 규제 골수성 세포 (RegMCs) 규정 하는 세포의 생체 내에서 세대 수 있습니다. 이 원고에서 식별 하 고 관용 유도 및 유지 관리. 에 그들의 책임을 확인 하기 위해 생체 외에서 그리고 vivo에서 규제 세포 활동을 정의 하는 데 사용 된 두 개의 보완 프로토콜 설명 첫째, 생체 외에서 진압 분석 결과 복용량 의존 방식에서 이펙터 면역 반응에 진압 용량 세포의 급속 한 식별을 허용 하 고 cytokine 측정 또는 세포 독성 등 추가 분석에 사용할 수 있습니다. 둘째, 새로 비친된 이식할된 받는 사람에 게 관용 대우 받는 사람에서 세포의 입양 전송 감독 이식 면역 반응 제어 및/또는 변환 하는 새로운 규정 하는 세포 (이러한 셀의 tolerogenic 속성을 강조 되 나 전염 성 관용). 이러한 방법은 알려진 phenotypic 표식이 셀에 제한 되지 않으며 모든 세포 인구를 확장할 수 있습니다. 또한, 기증자 감독 규제 셀 (필드에 중요 한 목표)의 allospecificity 제 3 자 기증자 세포를 사용 하 여 평가 될 수 있다 또는 이식 체 외에서 또는 vivo에서. 마지막으로,이 규정 하는 세포의 특정 tolerogenic 용량을 확인 하려면 우리 체액 항 기증자 항 체 반응과 새로운 또는 이전 알려진된 항 원에 대 한 체액 응답을 개발 하는 받는 사람의 용량을 평가 하기 위해 프로토콜을 제공 합니다. 설명 하는 관용의 모델 추가 규제 셀, 새로운 생체 및 immunoregulatory 분자 식별 하 하 사용할 수 있습니다 그리고 다른 이식 모델 또는 쥐 또는 인간 자기 면역 질병에 적응할 수 있다.

진압 활동 동시에 Apc (그림 1), 응답자 전지와 Tregs의 분류에 따라 (그림 2), 평가 또는 개별적으로 (그림 4), 그리고 어떤 다른 상 상속 규정 하는 세포 (그림 3), 수 완료 vivo에서 규정 하는 세포 및 생체 외에서 CFSE 밝기 (그림 5)의 측정에 의해 직접 주...

Watch this Scientific Journal Video about In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients at JoVE.com

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

여기, 선물이 프로토콜을 이식, 허용 오차를 유발 하 고 생체 외에서 그리고 vivo에서 평가 받는에서 고유한 셀 하위 집합의 진압 용량 및 기증자 또는 외 인 항 원 받는 사람의 면역 상태.

생체 외에서 그리고 Vivo에서 T, B 및 골수성 세포 진압 활동 및 이식 받는 체액 응답의 평가

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

in-vitro-vivo-assessment-t-b-myeloid-cells-suppressive-activity

Research

JoVE Journal

Immunology and Infection

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

14.1K 조회수.  INSERM, Université de Nantes. 여기, 선물이 프로토콜을 이식, 허용 오차를 유발 하 고 생체 외에서 그리고 vivo에서 평가 받는에서 고유한 셀 하위 집합의 진압 용량 및 기증자 또는 외 인 항 원 받는 사람의 면역 상태.

비디오: In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Murine B 세포를 정화에 유도 및 클래스 스위치 재조합 평가

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

연구

면역학 및 감염병학

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

기본 인간 T 세포 piggyBac Transposon 시스템 변경

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T 세포의 유도<em&gt; 체외</em&gt; 마우스 배아 줄기 세포에서

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

측정<em&gt; 체외</em&gt; HIV-1 사전 통합 단지의 통합 활동

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

분리는 차별화 및 정량화 인간의 혈액에서 B 세포를 항체가 분비 : ELISPOT을 체액 면역의 기능 판독으로

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

살 모 넬 라 typhimurium의 절연-대 식 세포에서 Phagosomes를 포함 하

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

인간 T 세포 회수 응답에서 Vivo에서 공부 하는 흡입 물집 프로토콜

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

정화 및 체 외에서 활동 분석 결과 (p) ppGpp Clostridium 남과 어울리지 않 는에서 합성에 대 한

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

생체 외에서 그리고 Vivo에서 T, B 및 골수성 세포 진압 활동 및 이식 받는 체액 응답의 평가

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