This research was supported by a grant from the NSERC Discovery program grant number RGPIN 418522-2013. We thank R. Kilgour for helpful discussions, and Katelin Gresty and Dr. A. Berghdal for providing the mouse muscle sections.

인간의 혈액 샘플을 연구 콩 코디 아 대학 윤리 심의위원회, 인증서 번호 10000618.에 의해 콩 코디 아 대학 윤리 심의위원회, 인증서 번호 2010BERG에 의해 승인 된 마우스 근육에 작업을 승인했다. 전혈 1. PBMC 분리 참고 : 멸균 기술과 제조 소독 장비를 사용하여 생물 안전 캐비닛에서이 절차를 수행합니다. <ol><li> 조심스럽게 멸균 50 ㎖ 원뿔형 튜브에 헤파린 (항 응집제) 혈액 수집 튜브에서 전혈 10-15 mL에 붓는다. 작은 혈액 유출시 청소 조직을 사용 DDH 2 O 70 % EtOH로 닦아냅니다. </li><li> 인산 버퍼 식염수 (PBS 1X) pH가 7.4 : 1의 비율로 1 원뿔 튜브에 혈액을 희석. 최대 총 용량은 30 ml를 초과하지 않는지 확인합니다. </li><li> 부드럽게 거품을 방지 혈청 학적 피펫 총을 사용하여 섞는다. 주 : 혈액을 방지하기 위해 튜브를 반전 혼합하지 마십시오원심 분리 동안 모자 및 후속 누출에 ccumulation. </li><li> 새로운 50 ㎖ 용량 원뿔 관에, 피콜 - 페이크 13 ml에 (재료 및 장비 표 참조)를 추가합니다. 랙에 튜브를 수직으로 유지합니다. </li><li> 전송 피펫을 사용하여, 희석 된 혈액을 상단에있는 튜브의 내부 벽에 전송 피펫 팁을 터치합니다. 느리고 일정한 압력으로 자당 층의 상부에 혈액 층을 형성하는 튜브의 내부 벽을 따라 혈액 옮긴다. 모든 혈액이 전송 될 때까지이 단계를 여러 번 수행합니다. </li><li> 단단히 튜브 씌우고 5 매체 가속도 세트로 스윙 로터 700 XG에 실온에서 30 분 동안 튜브를 원심 분리하고, 감속은 제로로 설정. </li><li> 천천히 튜브를 꺼내 레이어를 방해하지 않고, 바이오 안전성 캐비닛에 다시 걸릴. </li><li> 조심스럽게 PBS / 플라즈마 사이에 배치 된 PBMC이있는 버피 코트 (얇은 흐린 흰색 층), 수집차 피콜 층은 전송 피펫을 사용하여. 자당 층 수집 피하고 적혈구 층을 방해하지 않는다. </li><li> 새로운 50 ml의 멸균 원뿔 관에 버피 코트를 전송합니다. 그것은 모든 PBMC를 수집하는 단계 1.8을 반복 여러 번 걸릴 수 있습니다. </li><li> PBMC와 튜브에 PBS 산도 7.4을 추가하고 45 ml의 표시로 채워. 철저하게 튜브를 흔들어. 소용돌이하지 마십시오. </li><li> 워시 1 : 원심 분리기에서 15 분 동안 원추형 튜브의 하단의 한 PBMC 펠릿의 형성을 관찰 (9)에 설정된 최대 가속 및 감속 모두 480 XG에. </li><li> 표백제 10 ㎖와 플라스틱 비커에 PBS (상층 액)을 폐기하십시오. </li><li> 부드럽게 파상 표면 (즉, 빈 랙)에 대해 &quot;랙&quot;에 의해 세포 펠렛을 풉니 다. 소용돌이하지 마십시오. </li><li> 튜브에 신선한 PBS 산도 7.4의 25 ML을 추가합니다. 하나 이상의 튜브가 처리되는 경우,이 단계에서 함께 펠렛을 풀링. </li><li> 2 세척 : 원심 분리기에게 튜브를 12 분 동안 480 XG 위스콘신(9)에 설정된 최대 가속 및 감속 모두 토륨. </li><li> 표백제의 스플래시와 플라스틱 비커에 PBS (상층 액)을 폐기하십시오. </li><li> 파상 표면 (즉, 빈 랙)에 대한 조심스럽게 &quot;랙&quot;. 소용돌이하지 마십시오. </li><li> 튜브에 신선한 PBS의 25 ML을 추가합니다. <ol><li> 세포의 50 μl를 꺼내 생존 수에 대한 microcentrifuge 관에 전송합니다. </li><li> 트리 판 블루 (생존 얼룩)와 피펫 최대의 동일한 양 (50 μl를) 추가하고 아래로 부드럽게 혼합. </li></ol></li><li> 10 μl를 꺼내 1 ㎖ 당 세포의 생존 능력을 확인하기 위해 혈구로 전송. 세포 / ml의 수를 기록한다. </li><li> 세포의 원하는 금액을 가지고 9로 설정 최대 가속 및 감속 모두 480 XG에 12 분 동안 튜브를 원심 분리기. </li><li> 표백제와 비커에 PBS (상층 액)을 폐기하십시오. </li><li> 파상 표면 (즉, 빈 랙)에 대한 조심스럽게 &quot;랙&quot;. 8 추가튜브에 PBS의 0 μL. </li></ol> 2. PBMC 슬라이드 만들기 <ol><li> 현미경 슬라이드에 세포의 80 μl를 놓습니다. 다른 슬라이드의 도움으로 드롭 얼룩이나 슬라이드의 양쪽 끝에서 40 ㎕를 각 2 방울을 배치합니다. </li><li> 건조 할 수있는 생물 안전 캐비닛에 슬라이드를 둡니다. 프로스트 측에 연필로 슬라이드 레이블. </li></ol> 3. 슬라이드에 샘플을 고정 <ol><li> 4.5 ml의 99 % 에탄올 37 % 포름 알데히드 0.5 ㎖를 혼합하여 정착 솔루션을 준비합니다. </li><li> 슬라이드가 건조 된 후, 갓 정착액 용액 2 ㎖를 꺼내 슬라이드의 전체 표면이 피복되도록 슬라이드에 붓는다. </li><li> 1 분 동안 슬라이드에 솔루션을 남겨주세요. 수돗물로 1 분 동안 슬라이드를 씻어 공기 건조에 둡니다. </li></ol> 4. 음성 대조군의 아밀라아제 솔루션 만들기 <ol><li> 아밀라아제 분말 0.25 g을 디 50ml에 용해잠잠 물. 깨끗한 100 비이커에 용액을 붓고. </li><li> 슬라이드의 절반이 치료를 수신하고, 다른 절반은 미처리 유지 한 후, 실온 (도 1C)에서 15 분 동안 인큐베이션 비이커에 슬라이드 젖어. </li><li> 슬라이드의 측면 아밀라아제 치료를 받고있는 메모를합니다. 치료 및 제어 사이의 경계를 나타내는 슬라이드 뒷면에 선을 그립니다. </li><li> 아밀라아제 용액을 제거하고 공기 건조에 슬라이드를 떠나 DDH 2 O와 함께 슬라이드를 씻으십시오. </li></ol> 5. 요오드 산 쉬프 (PAS) 염색 및 이미지를 수행 주 : PAS는 시약 흡입 독성 및 부식성, 그래서 단계 화학 퓸 후드에서 수행해야하고, 노폐물을 적절히 기관 가이드 라인에 따라 처리되어야한다. <ol><li> 평평한 표면에 슬라이드를 삽입하고 샘플에 정기 산 용액의 1.50-2.00 ml에 붓는다. Incuba실온에서 5 분 동안 TE. </li><li> 증류수의 여러 비용이 슬라이드를 씻어. </li><li> 슬라이드에 쉬프의 시약의 1.50-2.00 ml에 붓고 실온에서 15 분 동안 그것을 품어. </li><li> 5 분 동안 증류수로 슬라이드를 세척하고 건조 공기에 둡니다. </li><li> 슬라이드에 10 μl의 설치 미디어를 적용하고 두 개의 작은 된 커버 커버, 또는 50 μl를 적용하고 하나의 큰 커버 슬립을 사용합니다. </li><li> 커버 슬립의 가장자리에 투명 매니큐어를 적용, 건조 하룻밤을 할 수 있습니다. </li></ol> 6. 100X 목표를 사용하여 쌍안 광학 현미경으로 이미지를 가져옵니다

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The authors have nothing to disclose.

이 비디오 문서의 중요한 단계는 세포의 세척 및 아밀라아제 치료 중이었다. 슬라이드를 세척하는 동안, 키 단계는 플라스틱 짤 세척 병을 사용하고 물이 부드럽게 슬라이드에 샘플을 통해 실행시키는 및 샘플에 직접 목표로하지 않았다. 조금이라도 물을 직접 압력은 슬라이드를 나서야 세포를 일으킬 것입니다. 또 다른 중요한 단계는 ± 아밀라아제 조건에 대해 동일한 슬라이드를 사용하는 것?...

요오드 산 쉬프 (PAS) 염색 널리 근육 연구 및 진단에 사용되는 면역 조직 화학 기술이다. 또한, 혈액 샘플에 대한 진단 도구로서 이용된다. 기술은 무색 쉬프 시약함으로써 진한 색 마젠타 생성물을 생산하는 반응 생성 다당류 알데히드기 유닛 내에 샘플을 산화에 요오드 산 용액을 적용하여 작동한다. 이 절차의 단계는 그림 1에 나타내었다. 얼룩은 글리코겐, 당 단백질, 당지질, 점액, 또는 다당류 부분과 다른 분자를 포함하는 다당류 마젠타, 아무것도를 켭니다. PAS 염색 종종 근육 섬유의 글리코겐 수준을 측정하기 위해 사용된다. 근육 조직 섹션은 단단히 슬라이드에 부착하고 여러 세척 및 염색 단계를 견딜로 기술에 이상적이다. 글리코겐은 높은 수요가 유형 II 근육 섬유, 빠른 트 위치에서 가장 존재최대 성능 1,2 글리코겐을 필요로하는 빠른 ATP 생산을위한. 글리코겐은 글리코겐 포스 포 릴라 제 효소의 작용을 통해 무료 포도당으로 분해 될 수있는 포도당의 분 지형 중합체이다. 휴식과 영양 자족의 시대에, 글리코겐, glycogenesis의 과정을 통해 보충하는 동안 영양 부족 또는 고 에너지 수요의 시대; 글리코겐은 글리코겐 분해에 의해 포도당으로 분해된다. 혈액 샘플에 이른 1950의 임상의 과학자들이 탐험으로 PAS 염색에서 각종 질병 3-7 글리코겐 함량을 분석합니다. 예를 들어, 폼 페병 질환-진실한 글리코겐 저장 질환 -에 백혈구 건강한 대조군 (8)로부터 크게 다르다 글리코겐 대량 축적된다. 이 비디오 문서에는 말초 혈 단핵 세포 (PBMC) 건강한 피험자에서의 정맥혈 샘플에 대한 사용 PAS 염색 적응 버전을 보여준다. PBMCS는 자연 살해 세포 및 단핵구와 같은 T 림프구와 B 림프구 가정뿐만 아니라 다른 면역 세포 림프구를 주로 함유한다. 제 정제 단계는 적혈구, 호중구 및 다른 과립구를 제거한다. 이 기술은 전체 혈액 도말하여 PAS에 비하여 양성 세포보다 강력한 열거 허용 림프구의 농축 비율에 데이터를 제공한다. <img alt="그림 1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> 도 1 :. PBMC에 PAS 염색 단계 방법에 의해 단계 (A) 우선, PBMC 단리는 피콜 구배 통해 이루어진다는, 왼쪽 패널 원심 분리 전에 제제를 도시 오른쪽 패널은 원심 분리 후를 도시 PBMC를 포함하는 버피 코트 관의 중심에서 관찰된다. (B)가 격리 된 PBMC 포르말린 - 에탄올 고착성을 사용 솔루 슬라이드에 고정되고기. 슬라이드 부드럽게 세척 플라스틱 병에서 증류수로 세정한다. (C)는 슬라이드 글리코겐 용해 아밀라제 용액으로 채워진 100 ㎖ 비이커 반쯤에 배치된다. 슬라이드 부드럽게 세정한다. (D)는 슬라이드 당류의 산화가 일어나는 요오드 산 용액으로 처리한다. 슬라이드 부드럽게 씻어 준다; 이는 과량의과 요오드 산을 제거하고 산화 공정을 중지한다. (E)을 쉬프 시약이 슬라이드에 첨가 될 때, 산화 단계 동안 만들어 알데히드와 반응 할 것이다. 이 무색 시약은 깊은 붉은 마젠타 제품에서 발생합니다. 슬라이드는 부드럽게 초과 쉬프 시약을 제거하기 위해 세척한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Periodic Acid Shiff Kit</td>
			<td>Sigma-Aldrich</td>
			<td>395B</td>
			<td>Bring to room temperature prior to use. Materials in this kit are toxic and harmful. Use caution&#160; 
			<a href="http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>&#945;-Amylase from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>A3176</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Binocular Microscope&#160;</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Axio Lab A0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Assay Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK016</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>VWR, GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Nonionic synthetic polymer of sucrose 
			<a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4779441">https://us.vwr.com/store/catalog/product.jsp?product_id=4779441</a></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For PBMC isolation, swing buckets were used&#160;</td>
		</tr>
	</tbody>
</table>

detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

시약 및 기본 기술의 유효성을 검증하기 위해, PAS 염색 마우스 가자미근 섹션에 제조업체의 지침에 따라 수행 하였다. 염색은 제물로 같은 날에 이루어졌다과 염색의 마지막 단계에서, 섹션 자일 렌에 의해 수정되었습니다. 가자미근은 ~ 35 % 글리코겐 양성 세포 구를 포함하는 것으로 알려져있다. 근육 세포 염색은 세포 내에서 두 개의 별개 PAS 양성 특징 - 점상 과립을 표시하고, 세포막 <s...

Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

detecting-glycogen-peripheral-blood-mononuclear-cells-with-periodic

Research

JoVE Journal

Immunology and Infection

21.1K 조회수.  Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University. Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

비디오: Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

마우스 복막 구멍 세포의 분리

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

연구

면역학 및 감염병학

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

공동 stimulatory 신호 봉쇄와 Alloantigen 자극에 의​​해 사람 말초혈 Mononuclear 세포 Alloantigen 특정 Anergy의 유도

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Zoledronate를 사용하는 사람 말초혈의 확장 γδ T 세포

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

를 사용하여 림프구 혈관 외로 유출 분석<em&gt; 체외</em&gt; 인간의 혈액 - 뇌 장벽의 모델

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.