Name: detection and enrichment of rare antigen-specific b cells for analysis of phenotype and function
Uploaded: 2017-02-16T15:00:00
Description: Watch this Scientific Journal Video about Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function at JoVE.com

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).

1. Isolation of Human PBMCs
<ol>
	<li>Collect 30-50 ml of blood using heparinized blood collection tubes. 
	NOTE: The amount of blood used depends on the particular experimental question and frequency in blood of antigen-specific B cells of interest. Heparinized blood can be processed immediately or rocked gently overnight at room temperature for processing the following day. The delay in processing has very little effect on the efficiency of enrichment and is associated with minimal loss of viability.</li>
	<li>M...

<ol>
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Here we describe a novel method to accomplish isolation and enrichment of antigen-binding B cells from human peripheral blood. The method is readily applicable to mice and to other tissues, such as the spleen and lymph nodes, and is compatible with post-enrichment analysis of cell phenotype and function (manuscript in preparation).
The user should be cognizant of a number of variables that can affect success of this procedure. From experience dead cells tend to stick to the magnetic beads and ...

Limiting dilution analyses of antibody-secreting cell precursor frequency have suggested that B cells reactive to a particular antigen typically occur at a frequency of 0.05 to 0.005% in the normal repertoire, depending on vaccination status and size/number of epitopes present on the antigen. The low frequency of these cells has made it difficult to study changes in their status during development of immune responses, such as following vaccination or exposure to a foreign antigen, or during development of autoimmunity. Previously, researchers have undertaken isolation of antigen-reactive B cells using techniques ranging from antigen coated plates or column adsorbents, to antigen-coated red blood cell resetting, to fluorescence-activated cell sorting1,2,3,4,5,6. Though these techniques have been successful in identifying and isolating antigen-reactive B cells, the results have varied in terms of yield, purity and scalability. Recently we developed a novel method to both detect and enrich rare B lymphocyte subpopulations using magnetic nanoparticles. The method enables enrichment with relatively high yield and purity from large starting populations, and is compatible with analysis of responses to antigen. By enriching from populations of cells in suspension, the method eliminates constraints that are associated with the geometry of antigen-coated plates or columns, and limit throughput. Finally, since enriched cells remain associated with antigen and a fluorescent reporter, they can be further purified by FACS sorting. As described herein we have used this approach for study of peripheral blood tetanus toxoid-reactive B cells before and following immunization of human subjects, as well as autoantigen-reactive B cells from subjects with various autoimmune disorders, including type 1 diabetes, Graves&#39; disease, and Hashimoto&#39;s disease7. The method works equally well in mouse and human, and is compatible with analysis of antigen-reactive B cells from a variety of tissues (manuscript in preparation).
In its basic format, peripheral blood mononuclear cells are first incubated with biotinylated antigen along with antibodies to cell surface antigens required for phenotypic analysis. This labeling step is followed by washing and fixation, and addition of streptavidin coupled to far-red-fluorescent dye for detection of the biotinylated-antigen binding cells (Figure 1). Previous studies have identified antigen-specific B cells in a similar manner but using antigens directly conjugated to a fluorochrome8,9,10,11. Although this is a worthy approach, use of biotinylated antigens in conjunction with streptavidin enables greater signal amplification (hence better differentiation of binding and non-binding cells), particularly when antigens are small12,13,14. An additional consideration is the use of streptavidin instead of avidin because streptavidin is deglycosylated, decreasing non-specific binding. Further, we use far-red-fluorescent dye as the fluorochrome due to its photostability, quantum yield (brightness), and its small size (~1.3 kD). Protein fluorochromes such as phycoerythrin (~250 kD) and allophycocyanin (~105 kD)15 are not optimal because they potentially contain many antigenic epitopes. Use of a small organic fluorescent dye composed of a single epitope, such as far-red-fluorescent dye, reduces complexity of the isolated cell population.
Once cells are biotinylated-antigen and far-red-fluorescent dye-streptavidin adsorbed, they are enriched using anti-far-red-fluorescent dye-conjugated magnetic nanoparticles. Single nanoparticles are not detected by most flow cytometers and therefore need not be removed prior to purification by FACS sorting and downstream assays16. Magnetic selection for antigen-specific B cells enriches the population of interest, eliminating the time and cost of sorting rare events using a flow cytometer.
Below we show representative results from enrichment of tetanus-toxoid-specific B cells from a subject before and seven days after tetanus toxoid booster immunization. We chose this particular application as an example in order to demonstrate the ability of this method to enrich antigen-specific B cells following acute in vivo stimulation. When coupled with flow cytometry, this method is capable of enriching and differentiating antigen-specific na&#239;ve, memory, and plasmablast B cells and allows the researcher to follow changes in their frequency over time. In addition, we include another possible downstream assay, e.g. an ELISPOT assay, which demonstrates that cells retain the ability to secrete antibody following enrichment. Another application of this method could involve adoptive transfer of enriched cells into a host. We have previously shown cells maintain the ability to act as antigen presenting cells to antigen-specific T cells following isolation and transfer (data not shown). Hence, there are a number of possible downstream assays that could be coupled to the method, which together informs the understanding of the antigen-specific immune response. We have described the method below, including controls to determine overall yield, purity, cell specificity, and fold-enrichment.

<table border="0" cellpadding="0" cellspacing="0" width="594">
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	<tbody>
		<tr height="99">
			<td height="99" style="height:99px;width:164px;">Antigen of interest</td>
			<td style="width:104px;">variable</td>
			<td style="width:131px;">variable</td>
			<td style="width:196px;">At least 100 &#956;g to biotinylate easily; if using protein try to use protein that has been validated by ELISA. It must be carrier&#160; protein free.</td>
		</tr>
		<tr height="118">
			<td height="118" style="height:118px;width:164px;">Biotin for labeling; e.g. EZ link Sulfo-NHS-LC-Biotin</td>
			<td style="width:104px;">e.g. Thermo Scientific</td>
			<td style="width:131px;">e.g. 21335</td>
			<td style="width:196px;">Biotin is available in different formulations, such as those containing&#160; various length spacers, so the type used should be determined by the researcher</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Streptavidin-Alexa Fluor 647</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:131px;">S21374</td>
			<td style="width:196px;">Can obtain from other suppliers.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:164px;">Anti-Cy5/Anti-Alexa Fluor 647 Microbeads</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">130-091-395</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:164px;">LS Columns</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">130-042-401</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">MACS manual separators</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">variable</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:164px;">Formaldehyde</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;">Dilute to 2% with PBS; optional if downstream assay requires live cells</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:164px;">PBS without calcium and magnesium</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:164px;">Ficoll-Paque PLUS</td>
			<td style="width:104px;">GE Healthcare</td>
			<td style="width:131px;">17-1440-02</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:164px;">Whole blood in heparinized collection tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:164px;">FACS buffer (PBS + 1% BSA + 0.01% sodium azide)</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:164px;">Separation buffer (PBS + 0.5% BSA + 2mM EDTA)</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:164px;">50 mL conical tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:164px;">15 mL conical tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:164px;">1.5 mL Eppendorf tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:164px;">Surface marker&#160; reactive antibodies, Fc Block, live/dead discriminating stain, if needed</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:164px;">ELISPOT supplies, if needed</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
	</tbody>
</table>

detection and enrichment of rare antigen-specific b cells for analysis of phenotype and function

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

Analysis of purity, yield, and fold-enrichment using flow cytometry
Populations enriched as described above inevitably contain contaminating cells that have not bound streptavidin- far-red-fluorescent dye but are trapped in the matrix. These impurities can be removed from enriched populations by FACS sorting. To estimate purity of enriched populations, gate on live cells based on forward and side scatter and/or live/dead stain and ...

Watch this Scientific Journal Video about Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function at JoVE.com

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

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

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

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