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 &#34;take up&#34; fluorescent dyes, such as the Streptavidin-far-red-fluorescent dye, leading to non-specific increased background. It is critical to remove as many dead cells from samples as possible before beginning this procedure. Use of stains that enable live/dead discrimination to gate out dead cells from the analysis may be helpful, but presence of dead cells may compromise interpretation of assays of antibody secreting cell frequency and function following adoptive transfer.
In addition, we have found that careful titration of the biotinylated antigen added is essential to distinguishing antigen-specific and non-specific binding. Use of too little antigen will lead to underestimation of the frequency of antigen-binding cells, while too much antigen can cause false positive detection/enrichment. Use of antigen concentrations that are too high may can be indicated by apparent binding to non-B cells in the samples. Further, apparent detection of an excessively high frequency (&#62;1%) of antigen reactive cells may be an indication of binding specificity issue. The ultimate goal when determining the optimal amount of antigen to use is to add a little bit more antigen than is present at physiological concentrations, such that the majority of the B cell receptors with an affinity of &#60;10-6 M, for example, for the antigen are saturated. This ensures that antigen-specific B cells are being detected with an affinity that has pathogenic potential. Addition of too little antigen, i.e. at or below physiological concentrations, may lead to detection of very few antigen-specific B cells due to decreased antigen occupation of the BCRs. On the other hand, addition of too much antigen, far exceeding physiological concentrations, can lead to increased non-specific binding which will include B cells with a low affinity for the antigen (&#62;10-6 M) and are actually ignorant of the antigen in vivo. Hence, it is vital to first titrate the biotinylated antigen across a few logs in order to be confident that only true antigen-reactive B cells are being detected. Similarly, we have found that use of too much streptavidin-far-red-fluorescent dye can increase background.
It is also important to note that biotinylation of antigen can affect availability of antigenic epitopes. Hence, optimization using different biotinylation methods may be important. Alternatively biotin can be coupled to primary amines, tertiary amines, sulfhydryls, or carboxyl groups. Use of extended spacer arm lengths can improve epitope availability by reducing steric hindrance.
One of the limitations to this technique is its reduced effectiveness for enrichment of antigen-binding B cells from cryopreserved compared to fresh cells. This could relate to greater cell death and associated &#34;stickiness&#34; in frozen samples. Nonetheless, the method can be applied to frozen samples but it is probably important not to directly compare results from enrichment and analysis of fresh and frozen cells. Another limitation is the purity of antigen-specific B cells in the enriched sample. From experience, there are always some cells that &#34;tag along&#34;, which includes both non-specific B cells and T cells. This is to be expected given the rarity of the cell population on is trying to enrich. However, the purity of the cells can be increase by ensuring that a single cell suspension is obtained before adding the cells to the column, enriching a second time on a new column, or by FACS sorting the antigen-specific B cells. Depending on the type of study, the researcher can decide which option is best and whether it is necessary.
As we optimized this technique we envisioned that it would be amenable to discrimination of high affinity versus low affinity antigen-reactive cells based on antigen titration or staining intensity. However, in a previous study, we showed antigen-reactive B cells with up to a 1,000-fold difference in affinity to their antigen were equally enriched using this method7. Hence, formal measurement of the affinity of enriched cells using surface plasmon resonance analysis of expressed recombinant antigen receptors is required if knowledge of the affinity is pertinent to the study.
Inherent in this technique is the limitation that addition of the biotinylated antigen and streptavidin can activate the cells by cross-linking the BCR. This could pose a problem for particular downstream functional assays, such as those that seek to compare stimulated cells to a resting control. This problem may be mitigated by keeping control samples cold to prevent transduction of signals. It is also possible that BCR signals could affect an ongoing biological response. From experience antigen-enriched cells maintain their ability to secrete antibody, as indicated by ELISPOT analysis (Figure 3b) and function as antigen presenting cells to T cells in vitro and after adoptive transfer into mice (data not shown). A final limitation to this technique is the use of anti-fluorochome antibodies to accomplish nanoparticle binding to antigen adsorbed cells. Anti- far-red-fluorescent dye cross-reacts with certain structurally related fluorochromes, including Cy7 and Cy5.5. This can limit the options of antibody conjugates available for measurement of phenotypic markers. However, with increased availability of new fluorochromes, this is becoming less of an issue.
Using this detection and enrichment method, it is possible to easily identify antigen-binding B cells in ex vivo peripheral blood, spleen, or other tissues. This method is particularly useful in studying the immune response of antigen reactive cells following immunization or exposure to foreign antigens and in the setting of autoimmune disease. Although no method to detect antigen-reactive B cells has proven to be without its faults, this method is fast, simple, and produces a relatively high purity, yield, and enrichment of antigen-reactive cells that can be coupled to a variety of downstream assays. In addition, due to the basic principles of the method, this procedure could easily be tailored to a variety of experimental needs.

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><tbody><tr><td>Antigen of interest</td><td>variable</td><td>variable</td><td>At least 100 &amp;mu;g to biotinylate easily; if using protein try to use protein that has been validated by ELISA. It must be carrier&amp;nbsp; protein free.</td></tr><tr><td>Biotin for labeling; &lt;em&gt;e.g.&lt;/em&gt; EZ link Sulfo-NHS-LC-Biotin</td><td>Thermo Scientific</td><td>21335</td><td>Biotin is available in different formulations, such as those containing&amp;nbsp; various length spacers, so the type used should be determined by the researcher</td></tr><tr><td>Streptavidin-Alexa Fluor 647</td><td>Invitrogen</td><td>S21374</td><td>Can obtain from other suppliers.</td></tr><tr><td>Anti-Cy5/Anti-Alexa Fluor 647 Microbeads</td><td>Miltenyi Biotech</td><td>130-091-395</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>MACS manual separators</td><td>Miltenyi Biotech</td><td>variable</td><td></td></tr><tr><td>Formaldehyde</td><td></td><td></td><td>Dilute to 2% with PBS; optional if downstream assay requires live cells</td></tr><tr><td>PBS without calcium and magnesium</td><td></td><td></td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>Whole blood in heparinized collection tubes</td><td></td><td></td><td></td></tr><tr><td>FACS buffer (PBS + 1% BSA + 0.01% sodium azide)</td><td></td><td></td><td></td></tr><tr><td>Separation buffer (PBS + 0.5% BSA + 2 mM EDTA)</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tubes</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tubes</td><td></td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>Surface marker reactive antibodies, Fc Block, live/dead discriminating stain, if needed</td><td></td><td></td><td></td></tr><tr><td>ELISPOT supplies, if needed</td><td></td><td></td><td></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 analyze far-red-fluorescent dye + cell frequency. The percentage within this latter gate is indicative of purity. Further analysis of the specificity and antigen affinity of these cells requires FACS sorting of single cells, followed by cloning and sequencing of expressed immunoglobulin genes and recombinant antibody production and analysis.
Yield refers to the number of isolated antigen-binding B cells recovered in Fraction A relative to the number in the starting population (Fraction D). Since the same number of cells were in Fraction D as Fraction A, one can directly determine the yield (# of antigen-binding B cells in Fraction A &#247; # of antigen-binding B cells in Fraction D).
Fold-enrichment (E) is reflected by the percentage of far-red-fluorescent dye + B cells in the &#34;positively&#34; enriched Fraction A relative to that in Fraction D. This value indicates the proportional increase in antigen-specific B cells achieved by nanoparticle enrichment. The value can be found using the following equation:
<img alt="Equation 1" src="/files/ftp_upload/55382/55382eq1.jpg" />
To demonstrate this technique, we show representative results obtained using tetanus-toxoid (Tet-Tox) as a model antigen to detect and enrich for Tet-Tox-reactive B cells in the peripheral blood of subjects who have been vaccinated against tetanus at varying time points. Figure 1 is a schematic of the method and the antigen adsorbent. Figure 2a shows the frequency of Tet-Tox-reactive B cells from an individual who was last vaccinated against tetanus more than a decade ago. Shown are the enriched (&#34;positive&#34;) and depleted (&#34;negative&#34;) populations from Fraction A and the baseline frequency of Tet-Tox-specific B cells from Fraction D. Hence, in this example we are able to enrich binding cells by around 7-fold and achieved a purity of 4% in the enriched fraction.
Figure 2b shows the specificity of the reaction using blood from a subject who had been vaccinated ~3 years earlier. When we added a 50-fold excess (50 &#181;M) of unlabeled tetanus-toxoid to the cells before addition of the biotinylated antigen (Fraction B) we are able to block binding by 83%, demonstrating the specificity of antigen binding. When we omitted biotinylated antigen from the procedure (Fraction C), a small number (~ 0.2%) of B cells still bound; this small fraction of B cells presumably reflects binding to some non-antigen in the adsorbant, such as the streptavidin&#8211;far-red-fluorescent dye, the anti&#8211;far-red-fluorescent dye antibody or the magnetic beads.
To demonstrate use of the method to analyze changes in antigen-binding cell phenotype following immunization we show in Figure 3 the frequency of Tet-Tox-specific B cells in the na&#239;ve, memory, and plasmablast populations in blood drawn before and 7 days after booster vaccination of a healthy subject. While the percentage of na&#239;ve B cells among total Tet-Tox-binding B cells decreased from 84% to 64% after boost, the percentage of memory and plasmablast populations among total Tet Tox-reactive cells increased from 16% to 36% and 0% to 40%, respectively. Figure 3b shows representative ELISPOT data from peripheral blood from the 7 days post-boost healthy subject. Isolation of the Tet-Tox-binding B cells led to 4-fold enrichment in tetanus toxoid antibody secreting-cell frequency.
<img alt="Figure 1" src="/files/ftp_upload/55382/55382fig1.jpg" /> 
Figure 1: Method for detection and enrichment of tetanus toxoid (Tet Tox)-binding B cells. (A) Protocol for enrichment and isolation of Tet-Tox-binding B cells. (B) Diagram of adsorbent employed to identify and enrich Tet-Tox-binding B cells. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55382/55382fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55382/55382fig2.jpg" /> 
Figure 2: Representative cytograms demonstrating enrichment of Tet Tox-binding B cells from human peripheral blood. (A) Cytograms of enriched and depleted Tet Tox-reactive B cells from Fraction A and total Tet-Tox-reactive B cells from Fraction D from a normal subject last vaccinated &#62;10 years previously. (B) Cytograms of PBMCs from the &#34;positive&#34; populations from a subject vaccinated ~3 years previously and enriched as in A (Fraction A), after pre-incubation with excess of 50 uM unlabeled Tet-tox (Fraction B), or enriched with Tet-Tox-biotin omitted during the procedure (Fraction C). All analyses were gated on CD19+ lymphocytes. Percentages represent the percent of Tet-Tox-binding cells of all B cells (CD19+) in the final fraction. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55382/55382fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55382/55382fig3.jpg" /> 
Figure 3: Examples of post-enrichment analysis of antigen-binding cells. (A) Representative flow cytometric analysis of cell phenotype following enrichment of Tet-Tox-binding B cells. Cytograms depict identification and analysis of Tet-Tox-reactive na&#239;ve (CD27-), memory (CD27+), and plasmablasts (CD27++ CD38++) in B cell subpopulations of a subject before and 7 days after booster vaccination against tetanus. All cells were gated on CD19+ lymphocytes from the enriched &#34;positive&#34; populations. (B) Representative ELISPOT analysis of antibody secreting cells in enriched, depleted, and total unenriched B cell fractions from peripheral blood of a healthy subject 7 days after boost. For the ELISPOT assay, wells of a 96 well plate were first coated with a 10 &#181;g/ml solution of tetanus-toxoid. Two-fold serial dilutions of cell suspensions were made in wells (across the plate) starting with cells at equal concentration from the enriched, depleted, and total unenriched fractions. Anti-tetanus antibody secreting cells were detected with biotinylated anti-human IgG (H&#38;L), followed by Streptavidin-AP. The plate was developed with ELISPOT development buffer and the reaction was stopped by washing the plate three times with double-distilled H2O. The number of spots at a cell dilution in the linear range was determined, and the number of ASCs was calculated. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55382/55382fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

detection-enrichment-rare-antigen-specific-b-cells-for-analysis

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

11.4K 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

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

Biology

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

Engineering

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

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 ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Bioengineering

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Isolation and Characterization of Antigen-Specific T Cells in Mouse Lungs

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Author Spotlight: Optimized Protocol for Detecting Antigen-Specific T Cells in Mouse Lungs Using Tetramers

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...