나는 면역 assays 및 마우스 처리 나를 소개하는 비앙카 Mothé, 칼라 Oseroff 및 마리 - 프랑스 DelGuercio 감사하고 싶습니다. GA 분배기의 연구실에서 작업이 NIH 그랜트 1 RO1 - AI - 073558, GLRCE 부여 1 - U54 - AI - 057153 및 바드 미국 3829-06에 의해 후원됩니다.

1. 예방 접종의 대상 - 특정 효과기 CTLs을 도출하는 것은 (주 0) <ol><li> 대상 시스템 : 시작하기 전에, 특정 이펙터에 따라 결정합니다. 생체내 CTL 분석의 고전이 예제에서, 우리는 특정한 CD8 + T 세포를 이끌어내는 수있는 정화 펩타이드 예방 접종을 사용합니다. 대상 시스템 : 동일한 펩타이드는 특정 이펙터를 작성, syngeneic 대상 세포를 펄스하는 데 사용됩니다. 또는 특정 effectors와 목표를 얻기 위해 전체 병원균이나 단백질을 사용하도록 선택할 수 있습니다. </li><li> 주사의 불임을 보장하기 위해 biosafety 후드의 작업 표면 안으로 오염을 제거하다. 먼저 두 ML 튜브에 포함된 PBS / 마우스 100 μL의 10 % DMSO에 펩타이드를 정화 50 μg을 배치하여 펩타이드 예방 접종을 준비합니다. 다음 혼합을 위해 1mm 구슬로 최대 0.2 ML 마크 매우 추운 불완전 프로 인드의 도움이되는의 동일한 금액을 추가합니다. 이 시점에서 사출까지 얼음에 immunogen 보관하십시오. Parafilm과 보안 덮개. </li><li> 즉시 혼합 후 얼음 다시 감광 유제를 삽입, 3 분 동안 감광 유제를 섞어 최대 RPM을 설정 구슬 때리는을 사용합니다. 결과 유제 일관성에 마요네즈와 유사해야합니다. 유제 4에 앉아 ° C 밤새 어떤 분리가 명시된 경우에는 시간이 허용하는가 (최대 주), 더 혼합하는 경우. 허용 </li><li> 모든 구슬을 끌어하지 않도록주의하고 유제를 잡아 1 ML의 주사기와 20 게이지 바늘 20.5을 사용하여 (구슬이 바늘의 측정보다 클 수 있지만, 여전히 인식해야합니다.) </li><li> 꼬리의 기지에 피하 주입을위한 보안 마우스. 우리는 안전에 대한 수정 플라스틱 50 ML 원뿔 관을 사용하여 부드럽게 주사 마우스를 고정. 유제의 0.2 ML로 마우스를 주사. </li></ol> 2. 수확 대상 셀 (주 8) <ol><li> 당신은 목표 세포에 대한 순진한 syngeneic 마우스가 필요합니다. AAALAC 기준에 따라 순진한 쥐를 안락사. </li><li> 마우스 머리 또는 기타 외부 오염 물질로부터 오염의 가능성을 최소화하기 위해 70 % 에탄올을 분사하여 마우스를 소독. </li><li> 해부 가위를 사용하면 마우스의 왼쪽에있는 피부를 통해 작은 싹둑합니다. 복막 색과 내장을 밝히기 위해서 피부 플랩을 세워. </li><li> 복막을 통해 싹둑, 조심되는 근본적인 기관 중 하나를 타협하지 않습니다. 비장은 표면에 가까이 있으며 컬러 간 (어두운 빨강)과 비슷하지만 작고 모양이 더 신장 콩입니다. 감염된 마우스는 일반적으로 감염되지 않은 컨트롤보다 특히 큰 spleens을 보여줍니다. </li><li> 조심스럽게있는만큼 최대 휴대폰 번호를 복구하기 위해서는 여러 조각으로 비장을 깰하지 부드럽게하는 간병, 비장을 당겨. 즉시 얼음 준비 매체 10 MLS를 포함한 15 ML 원뿔로 비장을 놓으십시오. 나머지 생쥐를 계속합니다. </li><li> 유리 조직 분쇄기로 15 ML 원뿔 튜브 (미디어 및 비장)의 내용을 하거라. 팔 위쪽 강도 많이 사용하여 비장을 분쇄. 모든 조직은 그것의 색깔을 잃었되면 당신은 결합 조직 밖으로 모든 세포를 회수했다고 가정 수 있습니다. </li><li> 50 ML 원뿔 관에 70 μm의 셀 필터를 통해 분쇄기의 내용을 하거라. 신선한 매체와 그라인더를 씻어 셀 스트레이너를 통해 부어. </li><li> 4 원심 분리기 ° C, 7 분 동안 1,300 RPM. 뜨는을 하거라. </li><li> 7분을 위해 37 °에서 준비 lysing 시약 및 부화 10 MLS에 Resuspend. </li><li> 신선한 미디어와 원심 분리기, 상기와 같은과 50 MLS 개까지 추가할 수 있습니다. </li><li> 50 MLS 신선한 미디어 및 원심 분리기 (세척 단계) 위와 같은에서 Resuspend. * 참고 : 순수한 대상 셀 인구가받는 마우스로 전송 원하는 경우이 시점에서 당신은 특정 세포 유형에 대한 자기 세포 분리 (예 : macrophages)를 수행할 수 있습니다. 또한이 시간에 당신은 특정 세포 죽이는 그 치료의 영향을 연구하기 위해 문화에 실험적 치료를 소개 수 있습니다. </li><li> 96 잘 라운드 바텀 조직 문화 처리 플레이트에 100μL의 X 10 7 잘 따라 세포 및 플레이트 1 카운트. *이 세포 / 음의 높은 농도이지만, 분석이 간단하기 때문에, 우리가이 농도에서 세포의 생존에 손실을 본 적이있다. </li></ol> 3. 대상 세포 치료 및 라벨링 <ol><li> 펄스 적절한 세포와​​ 병원체 특정 펩타이드 또는 펩타이드없는 컨트롤과 함께 복제 : 1 μg / ML 펩타이드를 추가합니다. 1.5 시간 4 ° C에서 알을 품다. 지난 30 분 동안 37 ° C 배양기로 이동합니다. 이 시간 동안 CFSE 시약을 준비합니다. </li><li> 제조 업체 사양에 따라 CFSE을 준비하고 0.5 및 25 μm의 사이에서 작동하는 농도로 희석. &quot;높음&quot;농도 CFSE보다 낮은 &#39;낮음&#39;농도가 10X합니다. 예를 들어, [낮은] 10 MLS PBS에 0.​​5 μL CFSE를 추가하고 위해 [고급] 10 MLS PBS로 5 μL CFSE를 추가합니다. 37 따뜻한 준비 CFSE ° C 레이블링하기 전에. </li><li> 인큐베이터와 P에서 세포의 플레이트를 제거합니다실온, 7 분 동안 1,300 rpm으로 고속 원심 분리기에 ellet. 뜨는 제거 플릭. </li><li> [낮은] 또는 해당 우물 적절한 시약 200 μL를 추가하고 37 15 분 동안 품어 °에서 [고급] CFSE C. 어느 Resuspend 세포 </li><li> 실온, 7 분 동안 1,300 rpm으로 보육하고 다시 펠릿 세포 플레이트를 제거합니다. 뜨는 제거 플릭. </li><li> 37 200 μL / 잘 예열 신선한 매체와 30 분 동안 품어의 Resuspend 세포 ° C. </li><li> 실온, 7 분 동안 1,300 rpm으로 보육 및 펠릿 세포 플레이트를 제거합니다. 뜨는 제거 플릭. </li><li> 100 μL / 잘 PBS에 Resuspend 세포. </li><li> 입양 전송을위한 하나 추가 물론 [낮은] 한 레이블 셀 잘 [고등] 마우스 세포마다 표시. 주사를 발생하는 0.2 MLS에 50% 구체적인 목표 50 % 관련이없는 세포가되어야합니다. </li><li> (그림 1 참조) 데이터 분석 도중 흐름 cytometer에서 실행에 대한 비 - 전송 제어의 역할을 몇 가지로 분류 세포를 별도로 설정하는 것을 잊지 마십시오. </li></ol> 4. 입양 전송 <ol><li> AAALAC 기준에 따라 이전 펩타이드 예방 접종을 쥐 (이 프로토콜의 일부로 # 1에서) 마취. </li><li> 받는 마우스로 역 궤도 경로를 통해 표시 대상 세포의 0.2 MLS를 주사. </li><li> 받는 사람 쥐를 euthanizing 6 시간 전의 또는 기타 미리 결정된 최적의 시간을 기다리십시오. </li></ol> 5. 수확 라벨 셀 * 참고 : 5.11 통해 5.1 2.11 통해 2.1 단계와 동일합니다 단계 <ol start="12"><li> 96 자 라운드 바텀 조직 문화 처리 플레이트에 100 μL의 X 10 6 자 당 1 셀 및 플레이트 아웃 카운트. </li><li> 모든 우물을 1 % paraformaldehyde의 최종 농도 100 μL 2 % paraformaldehyde를 추가하여 회수 및 비 전송 세포를 수정. </li></ol> 6. 유동세포계측법 및 데이터 분석 <ol><li> 흐름 cytometer에 샘플을 실행하고 데이터를 분석할 수 있습니다. </li></ol> 7. 대표 결과 결과 히스토그램에 떨어지 에피토프 특정 피크가 특정 세포 죽이는 (그림 1)의 지표이다. 특정 세포 용해의 실제 가치를 결정하기 위해 다음과 같은 방정식을 (표 1 적용)를 사용 : 비율 = 관련성이 비율 : 에피토프 특정 비율 ([낮은] 최대 : [고등] 피크), 퍼센트 특정 용해 = [1 - (비 전송 제어 비율 / 실험 비율)] X 100 <img src="/files/ftp_upload/2250/2250fig1.jpg" alt="그림 1" /> 그림 1. BALB / C 마우스 splenocytes를 발견 했어요. 숫자 CFSE 프로필은 높거나 낮은 CFSE의 표현형의 세포의 비율을 나타냅니다. A) 컨트롤을 비 - 양도. [낮은] CFSE의 봉우리가없는 펩타이드와 펄스 목표를 대표하는 동안 모든 [고급] CFSE의 봉우리는 에피토프 특정 타겟 세포를 나타냅니다. B) CFSE 라벨이 세포는 PBS 예방 접종을받는 마우스 (컨트롤)에서 복구. C) 두 개의 다른 펩타이드 특정받는 마우스에서 발견한 CFSE 라벨 세포는 감소 에피토프 특정 봉우리를 적어 둡니다. <img src="/files/ftp_upload/2250/2250fig2.jpg" alt="그림 2" /> 그림 2. 분석 데이터의 그래픽 표현. 쓰리 데이터 요소는 마우스의 각 그룹, PBS, 펩타이드 및 펩타이드 B.에 대한 표시 <table border="1"><tbody><tr><td> 견본 </td><td> CFSE HIGH %의 에피토프는 펄스 </td><td> 엉뚱 CFSE LOW % </td><td> 비율 낮음 : 높음 </td><td> 퍼센트 에피토프 특정 죽이는 </td></tr><tr><td> PBS </td><td> 42.8 </td><td> 57.2 </td><td> 1.34 </td><td> 16.97 </td></tr><tr><td> PBS </td><td> 43.1 </td><td> 56.9 </td><td> 1.32 </td><td> 15.94 </td></tr><tr><td> PBS </td><td> 44.3 </td><td> 55.7 </td><td> 1.26 </td><td> 11.74 </td></tr><tr><td> 펩타이드 </td><td> 39.7 </td><td> 60.3 </td><td> 1.52 </td><td> 32.56 </td></tr><tr><td> 펩타이드 </td><td> 38.6 </td><td> 61.4 </td><td> 1.59 </td><td> 35.61 </td></tr><tr><td> 펩타이드 </td><td> 40.6 </td><td> 59.4 </td><td> 1.46 </td><td> 29.99 </td></tr><tr><td> 펩타이드 B </td><td> 38.7 </td><td> 61.3 </td><td> 1.58 </td><td> 24.68 </td></tr><tr><td> 펩타이드 B </td><td> 38.5 </td><td> 61.5 </td><td> 1.60 </td><td> 25.32 </td></tr><tr><td> 펩타이드 B </td><td> 39.3 </td><td> 60.7 </td><td> 1.54 </td><td> 22.76 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> PBS NT 제어 </td><td> 47.4 </td><td> 52.6 </td&gt; <td> 1.11 </td><td></td></tr><tr><td> 응원 NT 제어 </td><td> 49.4 </td><td> 50.6 </td><td> 1.02 </td><td></td></tr><tr><td> 펩 B NT 제어 </td><td> 45.6 </td><td> 54.4 </td><td> 1.19 </td><td></td></tr></tbody></table> 표 1. 대표 데이터의 분석. &quot;NT&quot;는 아닌 전송됩니다.

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관심 없음 충돌 선언하지 않습니다. 동물 실험은 위스콘신 - 매디슨 대학에서 기관 애니멀 케어 및 사용위원회 (IACUC)에서 정한 지침과 규정에 따라 수행되었다.

CFSE가 자손 10,11 가운데 상대적으로 균등하게 분할하기 때문에 분석은 clonal 확장을 포함한 세포의 proliferative 용량을 조사하기 위해 수정할 수 있습니다. 이 예제 tetramers 또한 CFSE 라벨과 함께있을 수 있습니다 bioluminescence 13, 귀하의 가정에 따라 더 적절한 수 방법을 추적 기타 조직이있다하지만, 휴대 마이 그 레이션 6,12를 조사하기 위해 CFSE 라벨을 사용하는 것도 가능합?...

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<td>1 mm glass beads</td>
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<td>Vybrant CFSE Kit</td>
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<td>Invitrogen (Molecular Probes)</td>
<td>V12883</td>
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antigen specific in vivo killing assay using cfse labeled target cells

Carboxyfluorescein 디아 세 테이트 succinimidyl 에스테르 (CFSE)를 쉽고 빠르게 생체내 수사에 대한 관심의 세포 인구를 분류하는 데 사용할 수 있습니다. 이 라벨은 고전적인 증식 및 마이 그 레이션을 공부하기 위해 사용되었습니다. 여기서 제시하는 방법, 우리는 에피토프 특정 CFSE 표시 대상 세포 4-6의 생존과 살인 볼 입양 전송 후 일정을 단축했습니다. 그들의 수량이 오해 할 수 있으므로 CD8 + T 세포 클론의 특정 살인의 수준은 응답의 품질을 나타낼 수 있습니다. CD8 + T 특정 세포가 시토킨 생산 및 살인 7,8의 감소와 함께 시간이 지남에 따라 기능 소진 될 수 있습니다. 또한, 특정 CD8 + T 세포 클론은 TCR의 specificities 9 각기 다른뿐만 아니라 다른 사람으로 죽일 수 없습니다. 효과적인 세포 매개 내성 (CMI)의 경우, 항원은 T 세포 응답의 충분한 숫자뿐만 아니라 생산 파악뿐만 아니라, 강력한 기능 responding T 세포를해야합니다. 여기 BALB / C 마우스의 두 펩타이드 구체적인 T 세포 클론의 비율 세포 구체적인 살인을 평가합니다.

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

대부분의 감염은 강력한 CTL 반응을 이끌어내는,하지만 가끔 응답 세포의 수량은 병원균 제어 상관 관계를하지 않습니다<sup&gt; 1</sup&gt;. CTL 품질 측정 중 하나는 특별히 죽이는 능력이있다<sup&gt; 2</sup&gt;. 대상 세포의 CFSE 라벨이 CTL 응답 품질을 조사하는 데 사용할 수 있습니다<em&gt; 생체내에</em<sup&gt; 3,4</sup&gt;.

항원 특정 VIVO에서</emCFSE 라벨 대상 세포를 사용하여> 죽이는 분석

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

19.4K 조회수.  University of Wisconsin-Madison. 대부분의 감염은 강력한 CTL 반응을 이끌어내는,하지만 가끔 응답 세포의 수량은 병원균 제어 상관 관계를하지 않습니다 1. CTL 품질 측정 중 하나는 특별히 죽이는 능력이있다 2. 대상 세포의 CFSE 라벨이 CTL 응답 품질을 조사하는 데 사용할 수 있습니다 생체내에

비디오: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

연구

면역학 및 감염병학

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

효율적인위한 킬러 인공 항원 제시 세포 (KaAPC)<em&gt; 체외</em&gt; 인간의 항원 특이 T 세포의 고갈

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

자가 면역 대해 줄기 세포 유래의 항원 특이 적 조절 T 세포의 발달

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

표현형 및 기능 분석에 대한 검색 및 희귀의 농축 항원 특이 적 B 세포

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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

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

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

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

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

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

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

희귀 한 항 원 특정 B 세포 혈액 순환에서 구별 인간 단일 클론 항 체의 생성

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

항 원 특정 항 체 응답 다음 Clostridium 남과 어울리지 않 는 감염의 혈 청 학적인 결정에 대 한 단백질 Microarray 분석 결과

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic 살해 분석 결과 세균성 병원 체에 대 한 면역학 응답을 평가 하

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

카르복시플루아세인 수치니미딜 에스테르를 이용한 증식 인간 항원 특이적 CD4+ T 세포의 정량화

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

항원 특정 VIVO에서 죽이는 분석