我们感谢博士。罗伯特BONNEAU和尼尔·克里斯滕森有益的意见。 这项工作是由美国宾夕法尼亚州立大学医学院的资金支持。

1。从脾和淋巴结肿大BDC2.5小鼠的T细胞的分离<ol><li>使用6周龄糖尿病前期致糖尿病的CD4 + T细胞的捐助者的的女BDC2.5小鼠。应该是无糖尿病老鼠尿葡萄糖测定（见下文）所确定的。 </li><li>安乐死每个鼠标采用CO 2窒息，在无菌条件下取出脾脏，腋窝和肱淋巴结。要取出脾脏，用70％乙醇浸泡皮草，再切和缩回皮肤。脾将是可见的，为暗红色的器官上的左侧的鼠标。 1英寸腹膜切口用小剪刀，，轻轻抓住脾的中心有一对小镊子。小心地修剪的结缔组织和连接的脂肪尽可能地取出脾脏。 </li><li>收集10毫升Dulbecco&#39;s改性的Eagle培养基（DMEM）中，在冰上的15毫升锥形管中的脾脏和淋巴结。 </li><li>准备单细胞悬液通过使用日e结束了10ml无菌注射器的柱塞轻轻按压至70微米的细胞过滤器（脾每个过滤器和过滤器每4-6个淋巴结）同样的50毫升锥形管的淋巴器官。 </li></ol>在这个过程中，用1毫升的DMEM几次最大限度地回收细胞从过滤器冲洗每个过滤。 <ol start="5"><li>从50毫升锥形管中收集的细胞转移到15毫升锥形管，离心300〜400×g离心7分钟，在RT。 </li><li>以裂解红血细胞，弃去上清液，再使细胞悬浮在5ml二氯化铵钾（ACK）缓冲液（pH7.2），和孵化的ACK的细胞悬浮液在室温下5分钟。 </li><li>加入10毫升的DMEM到的ACK的细胞悬浮液，，如上离心管。一次10毫升的DMEM洗涤细胞沉淀。 </li></ol> 2。荧光激活细胞分选致糖尿病的CD4 + T细胞从BDC2.5老鼠<ol><li>重悬细胞在5毫升FACSŞ泰宁缓冲液，计数活细胞的数量（用相衬显微镜和血细胞计数器），用台盼蓝染料排斥。 </li><li>用FACS缓冲液中，调整至5×10 7个细胞/ ml的细胞悬液体积。删除〜1×10 6细胞每染色控制（1 3单无污点，污点）。染色样品的非转基因活化的CD4 + T细胞（APC）与抗CD4，抗TCRVβ4（FITC）和抗CD62L（PE）的单克隆抗体（单克隆抗体）在15毫升管。使用的单克隆抗体由生产商建议的浓度在4℃下20-30分钟，在黑暗中进行细胞染色。 </li><li>洗&gt; 3X染色反应体积的样品和单染色流式细胞仪缓冲控制。在300-400×g离心管，除去上清液，在FACS缓冲液中重悬细胞（1-2×10 7细胞/毫升样品进行排序和单染色法控制为300微升），并在冰上保存，直到下一个步骤。 </li><li>通过一个35微米的细胞，通过细胞样品滤网盖管，以去除细胞团块。排序样本的CD4 + TCRVβ4+ CD62L +细胞细胞分选仪与受过训练的操作员。收集3毫升的DMEM将细胞分成15毫升管中。允许2.5-3小时，包括设置，排序1.5×10 8个细胞（细胞的大致数量从3个供体小鼠）中获得某种率2×10共4个事件/秒。期待〜2.5×10 6转基因非激活的CD4 + T细胞的细胞分选后，每只小鼠。 </li><li>录制结束时的排序排序细胞的绝对数量和离心7分钟300-400 X G。弃上清，重悬细胞（2×10 6细胞/ ml），在无菌磷酸盐缓冲盐水（PBS）（毫克2 + / Ca 2 +的免费）收养T细胞转移。 </li></ol> 3。继转移致糖尿病的CD4 + T细胞从BDC2.5老鼠<ol><li>用1毫升注射器和18-1½针头，轻轻重悬流式细胞仪纯化的CD4 +T细胞和加载的1毫升注射器。在准备注射，更换18-1½针头与27½针头。 </li><li>直到他们表现出修饰行为暴露6-8周龄雌性NOD.SCID的收件人小鼠的加热灯。 </li><li>在限制器抑制受体小鼠，并用70％（V / V）乙醇消毒注射部位擦拭他们的尾巴。 </li><li>分为横向的尾静脉注射1-2×10 6流式细胞仪的排序条件（在500μl的PBS）细胞/小鼠。 </li></ol>不要迫使柱塞。如果针头适当地位于在静脉注射的地方几乎没有阻力。 4。监控受体小鼠高血糖和T1D <ol><li>开始5天后的T细胞转移，NOD.SCID受体小鼠每天监测尿糖升高使用试剂条（拜耳Diastix），根据制造商的说明。与两个连续urin老鼠ê血糖读数&gt; 250毫克/分升被认为是糖尿病患者。 </li></ol>

<ol>
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所有小鼠饲养在宾夕法尼亚州立大学医学院无特定病原体（SPF）设施的指引根据宾夕法尼亚州立大学的机构动物护理和使用委员会。 作者宣称，他们有没有竞争经济利益。

1型糖尿病可以诱发受体小鼠的整个糖尿病NOD小鼠或来自致糖尿病性T细胞克隆的TCR转基因小鼠的脾细胞或T细胞亚群的过继转移在不同的效率。我们在此报告可重复的方法，在两周内100％的发病率在受体小鼠诱发T1D通过流式细胞仪纯化CD62L + BDC2.5转基因的CD4 + T细胞的转移到NOD.SCID小鼠。 这里所描述的特定优势的BDC2.5 T细胞传输模型，包括诱导时间很短，相比T1D月自发糖尿病和糖...

NOD小鼠自发地开发自身免疫性糖尿病，并已被广泛用作人I型糖尿病的动物模型1,2。 I型糖尿病NOD小鼠的发病机制中的浸润，其特征在于，开始在3-4周的年龄，胰腺胰岛的树突状细胞和巨噬细胞，T细胞和B细胞。这一阶段的非破坏性的周边胰岛炎导致一个缓慢的，逐渐产生胰岛素的胰腺β细胞的破坏，从而导致明显的糖尿病，由4-6个月3岁。已转让4,5脾细胞，CD4 + 6,7或CD8 + 8,9糖尿病NOD小鼠T细胞介导糖尿病NOD小鼠免疫功能低下，表明胰岛反应性T细胞在1型糖尿病的发病机制中发挥了核心作用。根据不同的实验条件下，糖尿病受体小鼠发展缓慢，在几个星期内，在这些研究中。同样，来自不同的T细胞克隆，费时和昂贵的致糖尿病性T细胞的培养，已报告的几个星期后，转移到受体小鼠7,10介导糖尿病。随着转基因小鼠表达的TCR来自CD4或CD8限制的致糖尿病的T细胞克隆，一些实验室的可用性，其后已表明，从小鼠脾脏T细胞能够转移糖尿病收件人11-13。具体来说，NOD小鼠BDC2.5只转基因BDC2.5 TCR，这是具体 ​​的嗜铬粒蛋白A，胰腺β细胞中的一种蛋白质14-16。 在体外激活或者未激活的全部或分馏公然糖尿病或糖尿病前期BDC2.5糖尿病小鼠转移到新生儿或免疫缺陷的NOD小鼠在不同的效率11,17-19脾细 ​​胞的转移。 我们描述了一个简单的方法，利用纯化的转基因的CD4 + T细胞在高效率和consiste的受体小鼠诱导T1D从糖尿病前期BDC2.5小鼠NCY。从这些小鼠中分离大量的幼稚，胰岛抗原特异性CD4 + T细胞通过荧光激活细胞分类（FACS），CD4 + CD62L + T细胞表达的转基因TCRVβ4的链。纯化的转基因T细胞，然后转移无需激活到NOD.SCID的老鼠，缺乏功能性T细胞和B细胞和胰岛炎和糖尿病无20。受体小鼠进行监测尿糖浓度升高，表明I型糖尿病，迅速发展后两周内的T细胞转移。 相比之下致糖尿病异构特异性T细胞转移的其他方法，我们的协议，使用流式细胞仪排序CD4 + T细胞，几乎完全表达的致糖尿病BDC2.5的TCR。由于他们的同质性，只有少数转移T细胞（约1×10 6个细胞/鼠标）T1D发展迅速，需要2个星期内，在100％的发病率。我们的协议的另一个优势是，irradiation的受体小鼠是没有必要的，因为它是为一些其他的方法。这种方法的一个潜在的局限性，它并没有允许调查，CD4和CD8 + T细胞亚群，或特别是CD8 + T细胞在糖尿病的贡献。 所描述的协议将是有用的研究的T1D的快速发展，介导的幼稚的，单特异性CD4 + T细胞，以及治疗策略干预胰岛抗原特异性的Th细胞的归巢至靶器官。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&alpha;BDC 2.5, Tcr&beta;BDC 2.5)</td>
			<td>JAX</td>
			<td>004460</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NOD.SCID mice (NOD.CB17-Prkdcscid/J)</td>
			<td>JAX</td>
			<td>001303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
			<td>Themo Scientific</td>
			<td>SH30022.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bayer Diastix</td>
			<td>Fisher Scientific</td>
			<td>AM2803</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile surgical tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile small pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile large pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70 &mu;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 &mu;m cell strainer cap tubes</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium-Chloride-Potassium (ACK) buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2 in dH2O</td>
		</tr>
		<tr>
			<td>BD FACSFlowTM sheath fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS staining buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD4 (APC) mAb</td>
			<td>Biolegend</td>
			<td>1005616</td>
			<td>clone RM4-5</td>
		</tr>
		<tr>
			<td>Anti-TCR V&beta;4 (FITC) mAb</td>
			<td>BD Biosciences</td>
			<td>553365</td>
			<td>clone KT4</td>
		</tr>
		<tr>
			<td>Anti-CD62L (PE) mAb</td>
			<td>BD Biosciences</td>
			<td>553151</td>
			<td>clone MEL-14</td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>BD Biosciences</td>
			<td>&nbsp;</td>
			<td>e.g. BD FACSAria III</td>
		</tr>
		<tr>
			<td>Heat lamp</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18-1&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

accelerated type 1 diabetes induction in mice by adoptive transfer of diabetogenic cd4+ t cells

非肥胖糖尿病（NOD）小鼠自发地开发自身免疫性糖尿病12周龄后，是最广泛研究的动物模型，人类1型糖尿病（1型糖尿病）。照射的受体小鼠的细胞转移的研究已经建立了1型糖尿病的发病机制在这个模型中，T细胞的关键。我们描述一个简单的方法迅速诱发T1D继转移纯化，小学的CD4 + T细胞前糖尿病NOD小鼠胰岛特定的T细胞受体（TCR）BDC2.5到NOD.SCID受体小鼠的转基因。这种技术的主要优点是可以在同一天，不要求收件人照射型糖尿病的发病率很高，引起T细胞转移后2周内完成隔离和致糖尿病性T细胞过继转移。因此，研究可以继续以更快的速度比I型糖尿病的发病机理和治疗干预的方法依赖于异质性T细胞群或克隆来自糖尿病NOD小鼠。

我们的研究结果表明隔离的转基因BDC2.5细胞表达CD62L，这是家二级淋巴器官，如胰腺淋巴结T细胞的关键。我们的研究结果进一步证明了强有力的能力，这个单特异性T细胞的人口转移迅速和有效地T1D到NOD.SCID受体小鼠。 图2中所示的分离的致糖尿病CD4 + T细胞源自BDC2.5老鼠的。每股供体小鼠细胞分选前获得约5×10 7细胞汇集脾和淋巴结肿大。流式细胞仪排序之...

Watch this Scientific Journal Video about Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells at JoVE.com

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

我们提供了一个可重复的方法，诱导1型糖尿病（T1D）在小鼠胰岛抗原特异性的，初级的CD4 + T细胞过继转移在两个星期内。

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 ...

accelerated-type-1-diabetes-induction-mice-adoptive-transfer

Research

JoVE Journal

Immunology and Infection

16.6K 次观看.  Pennsylvania State University College of Medicine. 我们提供了一个可重复的方法，诱导1型糖尿病（T1D）在小鼠胰岛抗原特异性的，初级的CD4 + T细胞过继转移在两个星期内。

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

科研

免疫与感染

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

CD4的制备<sup&gt; +</sup&gt; T细胞通过显微镜分析GD3和GD2神经节苷脂膜表达的

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

高盐抗原表达树突状细胞的分离和采用转移

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

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.

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

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

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

针对 1 型糖尿病、乳糜泻和 COVID-19 的高通量多重筛查

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and ...

Methods Article

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