Name: in vitro and in vivo assessment of t, b and myeloid cells suppressive activity and humoral responses from transplant recipients
Uploaded: 2017-08-12T14:00:00.000+00:00
Duration: 18 min 48 s
Description: 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

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><tbody><tr><td>&lt;strong&gt;animals&lt;/strong&gt;</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,&amp;nbsp;</td></tr><tr><td>BN third party donor rats</td><td>Janvier Labs, France</td><td></td><td>8 weeks old,&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;name&lt;/strong&gt;</td><td>&lt;strong&gt;company&lt;/strong&gt;</td><td>&lt;strong&gt;catalogue number&lt;/strong&gt;</td><td>&lt;strong&gt;comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;reagents&lt;/strong&gt;</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-TCRab</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&amp;nbsp;Scientific Inc, USA</td><td></td><td>Phosphate Buffer Solution without calcium and magnesium,&amp;nbsp;</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>CellTrace&lt;sup&gt;TM&lt;/sup&gt; CFSE cell proliferation kit</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#C34554</td><td></td></tr><tr><td>RPMI 1640 medium 1X</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#31870-025</td><td></td></tr><tr><td>penicilline streptomycine</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#15140-122</td><td></td></tr><tr><td>Hepes Buffer</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#15630-056</td><td></td></tr><tr><td>non essential amino acids</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#11140-035</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fisher&amp;nbsp;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&amp;reg; 450 Cell</td><td>Thermo Fisher&amp;nbsp;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&amp;nbsp;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&#039;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&amp;micro;g (=1 vial) of CFDA SE (component A) in 90&amp;mu;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&amp;mu;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>&lt;strong&gt;name&lt;/strong&gt;</td><td>&lt;strong&gt;company&lt;/strong&gt;</td><td>&lt;strong&gt;catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;equipments&lt;/strong&gt;</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&amp;micro;m and 60&amp;micro;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>&amp;nbsp;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&amp;nbsp;</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&amp;nbsp;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 ...

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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

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

사람의 체외에서</em면역 불균형의 초기 국가의 인정을위한 심사 도구를> 억제

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

연구

면역학 및 감염병학

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

이식 - 대 - 호스트 질병의 유도 및<em&gt; 생체 내</emMHC 검색 손쥐 모델을 사용&gt; T 셀 모니터링

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

실시간 탐지의 체 외 종양 세포 Apoptosis에 CD8에 의해 유도 된+ T 세포 종양 침투 골수성 세포의 면역 진압 기능 연구

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

암 연구학

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

항원 특정 규제 트랜스 생체 지연 형 과민 분석

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

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

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

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

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

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

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

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 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

표현형 및 만성 림프 구성 맥락 수막염 바이러스에 감염된 마우스에서 고립 활성화 규제 T 세포의 기능 분석

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

이미징 유동 세포 계측법을 사용하여 T 세포 Alloreactivity의 측정

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

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

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

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

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

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

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

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

사람의 체외에서 억제

이식 - 대 - 호스트 질병의 유도 및

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

실시간 탐지의 체 외 종양 세포 Apoptosis에 CD8에 의해 유도 된⁺ T 세포 종양 침투 골수성 세포의 면역 진압 기능 연구

항원 특정 규제 트랜스 생체 지연 형 과민 분석

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

T 세포 반응의 평가를위한 형광 대상 배열의 사용

표현형 및 만성 림프 구성 맥락 수막염 바이러스에 감염된 마우스에서 고립 활성화 규제 T 세포의 기능 분석

이미징 유동 세포 계측법을 사용하여 T 세포 Alloreactivity의 측정

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