检测和罕见富集抗原特异性B细胞的表型和功能分析
Summary
描述了采用磁性纳米颗粒，以检测和富集的功能和表型分析抗原反应性B细胞中的简单而有效的方法。
Abstract
B cells reactive with a specific antigen usually occur at a frequency of <0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.
Introduction
限制稀释抗体分泌细胞前体的频率的分析已经表明，该反应的特定抗原的B细胞通常发生在0.05〜0.005％，在正常剧目的频率取决于接种的状态和存在于抗原表位的大小/数量。这些细胞的低频率，因此难以的免疫反应，如接种后或暴露于外来抗原或自身免疫的发展过程中发育过程研究在它们的状态的改变。先前，研究人员已经进行的使用的技术，从抗原包被的板或柱吸附剂，抗原包被的红细胞复位，对抗原反应性B细胞的分离荧光激活细胞分选1，2，3，4，5，6。 Thougħ这些技术已成功地鉴定和分离抗原反应性B细胞，结果在收率，纯度和可扩展性方面变化。最近我们开发了一种新的方法来检测二者并用的磁性纳米粒子丰富罕见的B淋巴细胞亚群。该方法能够富集具有相对高产量和纯度从大的起动种群，并与对抗原的反应分析兼容。通过从悬浮液中的细胞群富集，该方法消除了与抗原包被的板或列，并限制可以通过的几何结构相关的约束。最后，由于富集的细胞保持与抗原和荧光报道相关联的，它们可以被进一步通过FACS分选进行纯化。如本文所述，我们使用从与各种autoi受试者这种方法对于外周血破伤风类毒素反应性B前细胞的研究和人类受试者的免疫接种后，以及自身抗原反应的B细胞mmune疾病，包括1型糖尿病，格雷夫斯病，桥本氏病7。该方法同样适用于小鼠和人，并与从各种组织（手稿中制备）抗原反应性B细胞的分析相容。
在其基本形式，外周血单核细胞首先用抗体与细胞用于表型分析所需表面抗原的沿生物素化的抗原温育。此标记步骤之后是洗涤和固定，以及另外的链霉耦合到远红荧光染料用于检测生物素化的抗原结合的细胞（ 图1）。以前的研究已经确定了抗原特异性的B细胞以类似的方式，但使用抗原直接缀合到荧光染料8，9，10，11。虽然这是一个值得的做法，与链霉结合使用生物素标记抗原能够取得更大的信号放大（绑定和非绑定细胞从而更好的区分），特别是当抗原是小12，13，14。另外一个考虑是利用链霉代替抗生物素蛋白，因为链霉去糖基化，减少非特异性结合的。此外，我们用远红荧光染料作为荧光由于其光稳定性，量子产率（亮度），并且它的体积小（〜1.3 KD）。荧光蛋白，如藻红蛋白（〜250 KD）和别（〜105 KD）15不是最佳的，因为它们可能含有大量的抗原表位。单一表位组成，如远红荧光染料小的有机荧光染料的使用，减少了分离的细胞群的复杂性。
一旦细胞被生物素化一ntigen和远红色荧光染料的链霉亲和吸附，他们使用的是抗远红荧光染料缀合的磁性纳米粒子富集。单个纳米颗粒不被大多数流式细胞仪检测到的，因此，不必纯化之前通过FACS分选和下游测定16除去。抗原特异性B细胞的磁性选择丰富感兴趣的人口，消除了时间和排序使用流式细胞仪罕见的事件的成本。
下面，我们显示从破伤风类毒素特异性的B细胞的富集来自破伤风类毒素加强免疫后前七天受试者代表性结果。我们为了说明此方法的充实下列体内刺激急性抗原特异性B细胞的能力选择了这个特定的应用，例如，当用流式细胞仪结合，这种方法能够丰富和分化抗原特异性天真，内存，以及浆母B细胞和允许研究者跟随随时间在它们的频率的变化。此外，我们还包括另一种可能的下游分析， 例如 ELISPOT测定，这表明细胞保留分泌以下富集抗体的能力。该方法的另一个应用可能涉及富集细胞过继转移入宿主。我们以前曾表明细胞保持作为抗原呈递以下隔离和传输细胞抗原特异性T细胞的能力（数据未显示）。因此，有许多可能被耦合到所述的方法，其中一起通知抗原特异性免疫应答的理解可能下游测定。我们已经描述下面的方法，包括控制，以确定总收率，纯度，细胞特异性，和折叠式富集。
Protocol
Representative Results
Discussion
在这里，我们描述了一种新方法来完成分离和从人外周血抗原结合的B细胞的富集。该方法是容易适用于小鼠和其它组织，如脾脏和淋巴结肿大，是与细胞表型和功能（手稿准备中）的富集后分析兼容。
用户应该认识到许多的可能影响该过程的成功变量。从经验死细胞倾向于粘到磁性珠粒和“占用”荧光染料，如链霉亲和远红荧光染料，导致非特异性背景增加。关键是要开始?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the JDRF (1-2008-994, 27-2012-450) and the National Institutes of Health (R01DK096492-05, R21AI124488-01, T32OD012201, and F30OD021477).
Materials
| Antigen of interest | variable | variable | At least 100 μg to biotinylate easily; if using protein try to use protein that has been validated by ELISA. It must be carrier protein free. |
| Biotin for labeling; e.g. EZ link Sulfo-NHS-LC-Biotin | e.g. Thermo Scientific | e.g. 21335 | Biotin is available in different formulations, such as those containing various length spacers, so the type used should be determined by the researcher |
| Streptavidin-Alexa Fluor 647 | Invitrogen | S21374 | Can obtain from other suppliers. |
| Anti-Cy5/Anti-Alexa Fluor 647 Microbeads | Miltenyi Biotech | 130-091-395 | |
| LS Columns | Miltenyi Biotech | 130-042-401 | |
| MACS manual separators | Miltenyi Biotech | variable | |
| Formaldehyde | Dilute to 2% with PBS; optional if downstream assay requires live cells | ||
| PBS without calcium and magnesium | |||
| Ficoll-Paque PLUS | GE Healthcare | 17-1440-02 | |
| Whole blood in heparinized collection tubes | |||
| FACS buffer (PBS + 1% BSA + 0.01% sodium azide) | |||
| Separation buffer (PBS + 0.5% BSA + 2mM EDTA) | |||
| 50 mL conical tubes | |||
| 15 mL conical tubes | |||
| 1.5 mL Eppendorf tubes | |||
| Surface marker reactive antibodies, Fc Block, live/dead discriminating stain, if needed | |||
| ELISPOT supplies, if needed |
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