由美国国立卫生研究院（CA133085吉隆坡）和美国癌症协会的资助（RSG - 09 - 209 - 01 - TBG的吉隆坡）支持。

1。实验转移小鼠模型<ol><li>前一天的肿瘤细胞的注射剂，种子之一T75容量瓶中，用高达1 × 10 7 CMS4了10 mL的RPMI培养基中含有10％的血清，以获得快速增长的肿瘤细胞中的细胞。过夜孵育在37 ° C。 </li><li>在注射当天，去除培养基，用PBS冲洗细胞一次，然后用0.05％胰酶EDTA收获肿瘤细胞在37 ° C为5分钟。停止与10 mL的RPMI培养基中含有10％血清中反应。细胞转移到锥形管。 </li><li>分拆为3分钟，在室温下在Sorvall传奇RT离心机在1300 rpm的细胞。去除上清。重悬在10毫升的新鲜1X的HBSS洗细胞，然后降速再次重悬在以同样的方式。 </li><li>血球计数肿瘤细胞。在HBSS中稀释的细胞，所以，每次注射所需的细胞悬浮在总体积100μL。 </li><li>在温水中浸泡一个烧杯的尾静脉扩张，暖在6-8周龄BALB / c小鼠。鼠标放置在一个板凳上的尾静脉限位。使用microsyringe尾静脉注入100μL的肿瘤细胞。使用无菌技术操作，以避免感染。注射后，适用于注射部位轻微的压力，直到出血停止。 </li><li>返回鼠标笼子，使肿瘤生长所需的阶段。 </li></ol> 2。细胞毒性T淋巴细胞（CTL）的过继转移免疫<ol><li>在转移的当天，吸管上下以重悬纯化的细胞毒性T淋巴细胞，或CTL的准备，如文中所述，。所有的细胞从一个板块转移到15 mL锥形管，不要让音量超过超过2 / 3管。 </li><li>无菌巴斯德吸管插入锥管和奠定下细胞的淋巴细胞分离液，或LSM，直到总量接近14毫升。 </li><li>要小心，不要去打扰层。在室温下20分钟在2000转离心。不要使用制动快后慢，旋转转子。 </li><li>转移到一个新的15 mL锥形管的CTL。容积约10毫升加入HBSS洗。 </li><li>细胞计数。在室温下5分钟1300转离心。重悬在HBSS中所需的细胞密度注射，每次注射总量保持在100μL。 </li><li>前面看到的那样在这个视频中注入荷瘤小鼠的CTL，使用相同的尾部注射技术。 </li><li>返回鼠标笼子，让CTL的互动与肿瘤。老鼠通常牺牲的CTL治疗后14-21天进行分析。 </li></ol> 3。肺转移的可视化<ol><li>为了直观肺转移，一个牺牲的鼠标在其上的一个泡沫塑料板的背面。 PIN的腿，以确保通畅进入气管。喷雾用70％乙醇的腹侧方。 </li><li>从中旬腹部开始，用剪刀沿中线切，通过胸腔，向唾液腺。公开气管。使用镊子，以消除气管周围的组织。 </li><li>线程下面的气管200μL枪头。一只手握住一角，轻轻气管和远离身体。 </li><li>平台旋转180 °。使用50 ml注射器注入印度墨水肺部通过气管。完全与油墨的肺部膨胀，直到你感到强烈的抵触情绪。 </li><li>用剪刀剪开气管。肺部下一双镊子滑动，并解除他们的鼠标。简要1L烧杯中的水冲洗肺部。 </li><li>在化学通风柜，肺部转移到闪烁的玻璃小瓶含5毫升的Fekete的解决方案。几分钟后，肿瘤组织会出现白色上黑色的肺部结节。肿瘤，现在可以计算和存储在无限期的Fekete的解决方案。 </li></ol> 4。在可视化的肺代表转移<ol><li>在这里，从与乳腺癌细胞株4T1注入小鼠的肺部显示白色斑点，说明肿瘤。一个IRF8显性负突变K79E的转染4T1细胞注射的小鼠肿瘤细胞的增强的转移潜能。 </li><li>这些图像显示CTL过继转移免疫组织学疗效分析。注射用生理盐水荷瘤小鼠表现为多发性肿瘤6天后，黄色箭头（一）表示。另一方面，表现出与肿瘤特异性T细胞注入小鼠体内，减少了肿瘤（二）。 </li><li>在相同的实验，墨汁治疗提供了一个简单的方法来衡量CTL过继转移的疗效。在这里，白荷瘤肺肿瘤结节明显区别于他们已成功地进行CTL过继转移，结节计数肺部允许学位定量不可能与组织学。 </li></ol> 5。代表性的成果<img src="/files/ftp_upload/2077/2077fig1.jpg" alt="图1" /> 图1为实验肿瘤的转移和CTL的适应性转移免疫小鼠模型的试验计划。红色圆点表示肿瘤细胞和绿色圆点表示的CTL。 <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="图2" /> 图2中断IRF8功能增强对肿瘤细胞的转移潜能。稳定转染的小鼠乳腺癌细胞株4T1载体（4T1.Vector）或向量表示一个IRF8显性负突变K79E（4T1.IRF8K79E）（15，16）。鼠标横向尾静脉注射四到肿瘤细胞。荷瘤肺膨胀与印度墨水可视肿瘤结节。肿瘤结节很容易被看作是黑色的肺组织背景上的白色斑点。 <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="图3" /> 图3。CTL过继转移免疫组织学疗效分析。鼠标横向尾静脉注入小鼠肉瘤细胞株CMS4 - Met的第四。三天后，生理盐水（A）或肿瘤特异性T细胞（B）分别注入荷瘤小鼠。常规H＆E染色组织学染色CTL治疗后6天，肺部进行了分析。肿瘤结节是由箭头指示。 <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="图4" /> 图4。墨汁通胀的肿瘤结节的可视化。鼠标横向尾静脉注入小鼠肉瘤细胞株CMS4 - Met的第四。三天后，生理盐水（对照）或肿瘤特异性T细胞（+ CTL）分别注入荷瘤小鼠。的CTL治疗14天后，肺部进行了分析。白点，这是肿瘤结节，允许容易量化的CTL治疗的疗效。

<ol>
	<li>Ryan, M. H., Bristol, J. A., McDuffie, E., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+extensive+pulmonary+metastases+in+mice+by+adoptive+transfer+of+antigen-specific+CD8(+)+CTL+reactive+against+tumor+cells+expressing+a+naturally+occurring+rejection+epitope.">Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope.</a> J Immunol. 167 (8), 4286-4292 (2001).</li><li>Caldwell, S. A., Ryan, M. H., McDuffie, E., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fas/Fas+ligand+pathway+is+important+for+optimal+tumor+regression+in+a+mouse+model+of+CTL+adoptive+immunotherapy+of+experimental+CMS4+lung+metastases.">The Fas/Fas ligand pathway is important for optimal tumor regression in a mouse model of CTL adoptive immunotherapy of experimental CMS4 lung metastases.</a> J Immunol. 171 (5), 2402-2412 (2003).</li><li>Liu, K., Caldwell, S. A., Greeneltch, K. M., Yang, D., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTL+Adoptive+Immunotherapy+Concurrently+Mediates+Tumor+Regression+and+Tumor+Escape.">CTL Adoptive Immunotherapy Concurrently Mediates Tumor Regression and Tumor Escape.</a> J Immunol. 176 (6), 3374-3382 (2006).</li><li>Yang, D., Stewart, T. J., Smith, K. K., Georgi, D., Abrams, S. I., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+IFN-gammaR+in+association+with+loss+of+Fas+function+is+linked+to+tumor+progression.">Downregulation of IFN-gammaR in association with loss of Fas function is linked to tumor progression.</a> International journal of cancer. 122 (2), 350-362 (2008).</li><li>Pages, F., Berger, A., Camus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+memory+T+cells,+early+metastasis,+and+survival+in+colorectal+cancer.">Effector memory T cells, early metastasis, and survival in colorectal cancer.</a> N Engl J Med. 353 (25), 2654-2666 (2005).</li><li>Galon, J., Costes, A., Sanchez-Cabo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type,+density,+and+location+of+immune+cells+within+human+colorectal+tumors+predict+clinical+outcome.">Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.</a> Science. 313 (5795), 1960-194 (2006).</li><li>Strater, J., Hinz, U., Hasel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+CD95+expression+predisposes+for+recurrence+in+curatively+resected+colon+carcinoma:+clinical+evidence+for+immunoselection+and+CD95L+mediated+control+of+minimal+residual+disease.">Impaired CD95 expression predisposes for recurrence in curatively resected colon carcinoma: clinical evidence for immunoselection and CD95L mediated control of minimal residual disease.</a> Gut. 54 (5), 661-665 (2005).</li><li>Camus, M., Tosolini, M., Mlecnik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+intratumoral+immune+reaction+and+human+colorectal+cancer+recurrence.">Coordination of intratumoral immune reaction and human colorectal cancer recurrence.</a> Cancer research. 69 (6), 2685-2693 (2009).</li><li>Dudley, M. E., Wunderlich, J. R., Yang, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer+therapy+following+non-myeloablative+but+lymphodepleting+chemotherapy+for+the+treatment+of+patients+with+refractory+metastatic+melanoma.">Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma.</a> J Clin Oncol. 23 (10), 2346-2357 (2005).</li><li>Srivastava, M. K., Sinha, P., Clements, V. K., Rodriguez, P., Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells+inhibit+T-cell+activation+by+depleting+cystine+and+cysteine.+Cancer+research.">Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. Cancer research.</a> 70 (1), 68-77 (2010).</li><li>Nagaraj, S., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+escape+mechanism+governed+by+myeloid-derived+suppressor+cells.">Tumor escape mechanism governed by myeloid-derived suppressor cells.</a> Cancer research. 68 (8), 2561-2563 (2008).</li><li>Nguyen, D. X., Bos, P. D., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nature reviews. 9 (4), 274-284 (2009).</li><li>Heijstek, M. W., Kranenburg, O., Rinkes, B. o. r. e. l., H, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+colorectal+cancer+and+liver+metastases.">Mouse models of colorectal cancer and liver metastases.</a> Digestive surgery. 22 (1-2), 1-2 (2005).</li><li>Yang, D., Ud Din, N., Browning, D. D., Abrams, S. I., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+lymphotoxin+beta+receptor+with+tumor-specific+T+lymphocytes+for+tumor+regression.">Targeting lymphotoxin beta receptor with tumor-specific T lymphocytes for tumor regression.</a> Clin Cancer Res. 13 (17), 5202-5210 (2007).</li><li>Yang, D., Thangaraju, M., Browning, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN+Regulatory+Factor+8+Mediates+Apoptosis+in+Nonhemopoietic+Tumor+Cells+via+Regulation+of+Fas+Expression.">IFN Regulatory Factor 8 Mediates Apoptosis in Nonhemopoietic Tumor Cells via Regulation of Fas Expression.</a> J Immunol. 179 (7), 4775-4782 (2007).</li><li>Yang, D., Thangaraju, M., Greeneltch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+IFN+regulatory+factor+8+by+DNA+methylation+is+a+molecular+determinant+of+apoptotic+resistance+and+metastatic+phenotype+in+metastatic+tumor+cells.">Repression of IFN regulatory factor 8 by DNA methylation is a molecular determinant of apoptotic resistance and metastatic phenotype in metastatic tumor cells.</a> Cancer research. 67 (7), 3301-3309 (2007).</li><li>Wexler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+identification+of+experimental+pulmonary+metastases.">Accurate identification of experimental pulmonary metastases.</a> Journal of the National Cancer Institute. 36 (4), 641-645 (1966).</li></ol>

小鼠购自美国国家癌症研究所（Friderick，MD）和住在医学院格鲁吉亚动物设施。实验和关怀/福利协议联邦法规和医学院格鲁吉亚IACUC委员会批准的协议。

解决方案: 印度国内的油墨解决方案（17）: <ol><li>倒入150毫升蒸馏水250毫升容量瓶中。 </li><li>加入4滴氢氧化铵的蒸馏水。 </li><li>氨和水的混合物中加入30毫升墨汁的股票（即桑福德魔法防水绘图墨水4465项44011）。 </li><li>顶用蒸馏水到200毫升的量。解决方案是准备注射。 </li></ol>的Fekete的解决方案（17）: 是用来漂白印度虚增墨荷瘤肺的Fekete的解决方案，以区别于正常组织的黑色背景白色的肿瘤结节。 <ol><li> 350毫升95％的乙醇添加1L玻璃瓶。 </li><li>添加150毫升蒸馏水</li><li>添加50毫升甲醛</li><li>添加25毫升冰川酸的酸性物质</li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

实验转移小鼠模型是一个简单的，但有关生理转移模型。肿瘤细胞静脉注射（四）为小鼠尾静脉和殖民，从而在肺部，类似肿瘤细胞自发转移的最后步骤:在流通领域，外渗及远端器官的殖民化的生存。从治疗的观点来看，实验转移模型是最简单和理想的模式，自疗法的目标往往是:建立在远端器官的转移瘤的转移终点。在此模型中，肿瘤细胞注射四到小鼠尾静脉，并允许在肺部进行殖民和增长。然后注入肿瘤特异性CTL转移轴承鼠标第四。肺转移的数量和大小，可以控制被注入肿瘤细胞的数量和肿瘤生长的时间。因此，各个阶段的转移，从最小的转移到全身广泛转移，可为蓝本。肺转移，从而允许更容易直观地观察和定量分析与油墨的通货膨胀。

Watch this Scientific Journal Video about Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model at JoVE.com

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

一个实验性肺转移和CTL免疫小鼠模型的肿瘤细胞- T细胞相互作用分析<em&gt;在体内</em&gt;。

实验转移和CTL过继转移免疫小鼠模型

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

experimental-metastasis-ctl-adoptive-transfer-immunotherapy-mouse

Research

JoVE Journal

Immunology and Infection

19.3K 次观看.  Medical College of Georgia. 一个实验性肺转移和CTL免疫小鼠模型的肿瘤细胞- T细胞相互作用分析在体内。

视频: Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

科研

免疫与感染

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

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

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

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

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

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

淋巴免疫治疗和疫苗接种小鼠

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

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

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

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

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

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

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

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

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

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

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

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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

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

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

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

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

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

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

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

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

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

新生儿多药脓毒症的对照小鼠模型

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

建立与艾滋病毒相关的肝脏发病机制的双人性化TK-NOG小鼠模型

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...