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>Mix whole blood 1:1 with sterile, room temperature magnesium and calcium free phosphate-buffered saline (PBS).</li>
	<li>In a 50 ml sterile conical tube, add 10 ml of room temperature density gradient solution. Store the opened density gradient bottles in the dark at 4 &#176;C, but warm to room temperature before use.</li>
	<li>If possible set the pipette gun to the slow setting and/or apply very little, yet consistent, trigger pressure to slowly layer 30-35 ml of the diluted blood on top of the density gradient, being careful not to mix the two. 
	NOTE: Placing the tip of the pipette against the edge of the 50 ml conical while adding the diluted blood will help ensure a consistent steady flow.</li>
	<li>Centrifuge at 400 x g for 30 min at 20 &#176;C with the brake turned off.</li>
	<li>Remove the upper layer, which contains plasma and platelets, being careful not to disturb the mononuclear cell layer below.</li>
	<li>Using a sterile pipette, collect the mononuclear cell layer (buffy coat) and place into a separate sterile 50 ml conical tube.</li>
	<li>Add PBS to the cell suspension to a volume of 50 ml. Centrifuge at 400 x g for 10 min at 20 &#176;C with brake.</li>
	<li>Remove supernatant and resuspend pelleted cells in 10 ml of PBS. Count cells and determine viability using a hemocytometer or automated cell counter.</li>
	<li>Resuspend cells at 107 cells/ml and place on ice until ready for further processing.</li>
</ol>
2. Staining of Cells
<ol>
	<li>Remove 1-2 x 106 cells for each fluorescence compensation/FMO control needed, if applicable. Centrifuge remainder of cells at 400 x g for 10 min at 4 &#176;C with brake.</li>
	<li>Resuspend cells in sterile filtered, cold FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.01% sodium azide) at 3-6 x 107 cells/ml (remove in a 1.5 ml centrifuge tube). Ensure that the FACS buffer is cold and keep it on ice throughout the staining procedure.
	<ol>
		<li>Divide the cell population into fourths (A-D) when optimizing the assay. Use fraction A to enrich antigen-specific B cells of interest, use fractions B and C to determine the specificity of enrichment/staining (see 2.5 and 2.6 below), and use fraction D to determine the frequency and number of antigen-specific cells in the unenriched population to enable calculation of yield and fold-enrichment.</li>
	</ol>
	</li>
	<li>Add human Fc&#947;R blocking reagent to fractions A-D on ice to prevent binding of antibodies to Fc receptors. 
	NOTE: Follow manufacturer&#39;s instructions for appropriate conditions.</li>
	<li>Add fluorochrome-conjugated antibodies to cell surface antigens of interest to fractions A-D for 30 min on ice in the dark, being careful not to include fluorochromes that may cross react with the anti-far-red-fluorescent dye antibodies coupled to magnetic nanoparticles. 
	NOTE: These include antibody conjugates to near-IR fluorescent dye, Cy5, Cy5.5, and Cy7. Any viability stains that are added should be compatible with the use of a fixative. If one does not intend to analyze the cells using FACS, but determine the antibody-secreting frequency, for example, by ELISPOT, surface staining with antibodies is unnecessary.</li>
	<li>To test the specificity of the assay, incubate Fraction B with sufficient amount of unlabeled antigen for 30 min on ice, such that the majority of the receptors with an affinity of at least 10-6 M are blocked. 
	NOTE: Typically, a good starting concentration is a 50-100 fold excess of the amount of antigen deemed sufficient in 2.6 below (e.g. 50-100 &#181;M). This should block any B cells with a high affinity for the unlabeled antigen to bind to the biotinylated antigen, which is added in step 2.6 below, thus allowing confirmation of the specificity of the cells enriched. This step can be completed in conjunction with step 2.4 above.</li>
	<li>Add biotinylated antigen to Fractions A, B, and D. Incubate on ice for 30 min. 
	NOTE: Concentration of biotinylated antigen should be titrated to determine the optimal amount needed to ensure adequate detection and separation of binding cells from non-specific bystander cells. Typically a good starting concentration to test is 1-5 &#181;M. 
	NOTE: For Fractions A and D, this step can be done concurrently with step 2.4 above. For Fraction B, this step should be done after step 2.5 (washing in between steps is unnecessary).</li>
	<li>Omit biotinylated antigen from Fraction C in order to determine any binding of B cells to other reagents in the protocol, such as streptavidin- far-red-fluorescent dye and the magnetic beads.</li>
	<li>Wash cells twice by suspension of cells in 1 ml of cold FACS buffer per 5 x 107 cells and centrifugation at 400 x g for 5 min at 4 &#176;C.</li>
	<li>After the final wash, dilute concentrated cell suspension in 1 ml 2% formaldehyde per 5 x 107 cells and let sit in dark on ice for 5 min. 
	NOTE: Fixation of the cells at this point ensures that the biotinylated antigen remains bound to the BCR for the remaining of the procedure. For viable cells &#8211; for downstream analysis, such as in ELISPOTs or adoptive transfers, omit the fixation step.</li>
	<li>Wash cells twice with 1 ml of cold FACS buffer per 5 x 107 cells and centrifuge at 400 x g for 5 min at 4 &#176;C. Resuspend in 1 ml cold FACS buffer per 5 x 107 cells.</li>
	<li>Add 1-2 &#181;g streptavidin-far-red-fluorescent dye/ml and incubate on ice for 20 min in the dark. Titrate the amount of streptavidin for each application.</li>
	<li>Wash cells twice with 1 ml cold FACS buffer per 5 x 107 cells and centrifuge at 400 x g for 5 min at 4 &#176;C. Fraction D preparation is completed at this point as it will not undergo enrichment using the magnetic beads. 
	NOTE: This sample will be used to determine the frequency and number of antigen-specific B cells in the sample. This will be used to determine the yield and fold-enrichment achieved by enrichment.</li>
</ol>
3. Magnetic Nanoparticle-based Enrichment
<ol>
	<li>Resuspend cells from samples A-C in 1 ml of cold separation buffer (PBS + 0.5% BSA + 2mM EDTA) per 5 x 107 cells and pass through a 40 &#181;m filter to eliminate any clumps that could clog the column.</li>
	<li>Add Anti-Cy5/Anti-far-red-fluorescent dye nanoparticles. For optimal results, the concentration of nanoparticles should be titrated for each use, but a good starting point is 50 &#181;l suspension per 5 x 107 cells/ml. Rotate in the dark at 4 &#176;C for 10 min.</li>
	<li>Wash twice with 1 ml cold separation buffer at 400 x g for 5 min at 4 &#176;C. Resuspend cells in 1 ml of cold separation buffer per 5 x 107 cells.</li>
</ol>
4. Magnetic Enrichment Using LS or LD Columns
<ol>
	<li>Place three LS or LD columns in the magnetic field of a magnetic separator and add 3 ml of separation buffer to wet the columns (see product description to determine which type of column is best suited). Allow all 3 ml to pass through the column and discard after collection. 
	NOTE: Although separation of magnetic nanoparticle labeled cells can be accomplished using any one of the magnetic separation devices that are commercially available, the laboratory has had the most success with the use of LS columns, in terms of yield, purity, and efficiency of enrichment.</li>
	<li>Place three 15 ml conical tubes labeled &#34;Fraction A/(B)/(C) negative&#34;, for negatively selected cells, under the column. Add the magnetic particle labeled cells to their respective column and allow the entire volume to pass through.</li>
	<li>Add 3 ml of cold separation buffer on top, and allow this to pass through the column and repeat with 2 ml. Collect the negatively selected cells and set aside on ice.</li>
	<li>Remove the column from the magnetic field and place on top of a 15 ml conical tube labeled &#34;Fraction A/(B)/(C) positive&#34;, for positively selected cells.</li>
	<li>Fill the column to the top with approximately 6 ml of separation buffer and immediately plunge this volume through the column to collect cells that had bound to the magnetic field using the provided plunger.</li>
	<li>To increase purity, the positively selected cells can be further enriched using a second clean column, repeating the procedure.</li>
	<li>Spin both the negatively and positively selected fractions at 400 x g for 10 min at 4 &#176;C and suspend in desired final volume. Proceed with downstream analysis, such as FACS, ELISPOT or adoptive transfer.</li>
</ol>

<ol>
	<li>de Wildt, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+analysis+and+production+of+monoclonal+antibody+fragments+originating+from+single+human+B+cells.">A new method for the analysis and production of monoclonal antibody fragments originating from single human B cells.</a> J Immunol Methods. 207 (1), 61-67 (1997).</li><li>Edelman, G. M., Rutishauser, U., Millette, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fractionation+and+arrangement+on+fibers,+beads,+and+surfaces.">Cell fractionation and arrangement on fibers, beads, and surfaces.</a> Proc Natl Acad Sci U S A. 68 (9), 2153-2157 (1971).</li><li>Haas, W., Schrader, J. W., Szenberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new,+simple+method+for+the+preparation+of+lymphocytes+bearing+specific+receptors.">A new, simple method for the preparation of lymphocytes bearing specific receptors.</a> Eur J Immunol. 4 (8), 565-570 (1974).</li><li>Kenny, J. J., Merrill, J. E., Ashman, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-step+centrifugation+procedure+for+the+purification+of+sheep+erythrocyte+antigen-binding+cells.">A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.</a> J Immunol. 120 (4), 1233-1239 (1978).</li><li>Snow, E. C., Vitetta, E. S., Uhr, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+antigen-enriched+b+cells.+I.+Purification+and+response+to+thymus-independent+antigens.">Activation of antigen-enriched b cells. I. Purification and response to thymus-independent antigens.</a> J Immunol. 130 (2), 607-613 (1983).</li><li>Egeland, T., Hovdenes, A., Lea, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+of+antigen-specific+B+lymphocytes+by+means+of+immunomagnetic+particles.">Positive selection of antigen-specific B lymphocytes by means of immunomagnetic particles.</a> Scand J Immunol. 27 (4), 439-444 (1988).</li><li>Smith, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+anergic+B+cells+in+prediabetic+and+new-onset+type+1+diabetic+patients.">Loss of anergic B cells in prediabetic and new-onset type 1 diabetic patients.</a> Diabetes. 64 (5), 1703-1712 (2015).</li><li>Hulbert, C., Riseili, B., Rojas, M., Thomas, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+specificity+contributes+to+the+outcome+of+diabetes+in+nonobese+diabetic+mice.">B cell specificity contributes to the outcome of diabetes in nonobese diabetic mice.</a> J Immunol. 167 (10), 5535-5538 (2001).</li><li>Woda, M., Mathew, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescently+labeled+dengue+viruses+as+probes+to+identify+antigen-specific+memory+B+cells+by+multiparametric+flow+cytometry.">Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry.</a> J Immunol Methods. 416, 167-177 (2015).</li><li>Nair, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+fluorescent+labeling+to+identify+memory+B+cells+specific+for+Neisseria+meningitidis+serogroup+B+vaccine+antigens+ex+vivo.">Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo.</a> Immun Inflamm Dis. 1 (1), 3-13 (2013).</li><li>Amanna, I. J., Slifka, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+rare+memory+B+cell+populations+by+two+independent+and+complementary+approaches.">Quantitation of rare memory B cell populations by two independent and complementary approaches.</a> Journal of immunological methods. 317 (1-2), 175-185 (2006).</li><li>Scheid, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+identification+of+HIV+gp140+binding+memory+B+cells+in+human+blood.">A method for identification of HIV gp140 binding memory B cells in human blood.</a> Journal of immunological methods. 343 (2), 65-67 (2009).</li><li>Doucett, V. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+and+characterization+of+virus-specific+B+cells+by+multicolor+flow+cytometry.">Enumeration and characterization of virus-specific B cells by multicolor flow cytometry.</a> Journal of immunological methods. 303 (1-2), 40-52 (2005).</li><li>Malkiel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Checkpoints+for+Autoreactive+B+Cells+in+Peripheral+Blood+of+Lupus+Patients+Assessed+By+Flow+Cytometry.">Checkpoints for Autoreactive B Cells in Peripheral Blood of Lupus Patients Assessed By Flow Cytometry.</a> Arthritis Rheumatol. , (2016).</li><li>Pape, K. A., Taylor, J. J., Maul, R. W., Gearhart, P. J., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+B+cell+populations+mediate+early+and+late+memory+during+an+endogenous+immune+response.">Different B cell populations mediate early and late memory during an endogenous immune response.</a> Science. 331 (6021), 1203-1207 (2011).</li><li>Abts, H., Emmerich, M., Miltenyi, S., Radbruch, A., Tesch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD20+positive+human+B+lymphocytes+separated+with+the+magnetic+cell+sorter+(MACS)+can+be+induced+to+proliferation+and+antibody+secretion+in+vitro.">CD20 positive human B lymphocytes separated with the magnetic cell sorter (MACS) can be induced to proliferation and antibody secretion in vitro.</a> Journal of immunological methods. 125 (1-2), 19-28 (1989).</li></ol>

The authors declare they have no competing financial interests.

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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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Research

JoVE Journal

Immunology and Infection

11.3K Views.  University of Colorado School of Medicine. A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...