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

This work was supported by grants from the JDRF (1-2008-994, 27-2012-450) and the National Institutes of Health (R01DK096492-05, R21AI124488-01, T32OD012201, and F30OD021477).

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

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

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

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

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

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

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

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

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

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

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

The overall goal of this simple yet effective procedure is to isolate and enrich for rare antigen-binding B cells from human peripheral blood using magnetic nanoparticles for their phenotypic and functional analysis. This method can help answer key questions in the field of immunology regarding the phenotype and biology of rare antigen-binding B cells both before and following in-vivo antigen encounter. The major advantage of this technique is that it obviates problems associated with geometry, particularly when one is using a styrene plate or a large bead.And it obviates problems associated with flow rate when one is isolating cells by flow cytometry to isolate rare cells in quite large starting cell populations. Immediately after collecting 30 to 50 milliliters of whole blood into heparinized blood collection tubes, mix the blood at a 1:1 ratio with sterile room-temperature magnesium and calcium-free PBS. Next, layer the blood mixture onto 10 milliliters of room-temperature density gradient solution in a sterile 50 milliliter conical tube using slow consistent pipette trigger pressure.Separate the cells by centrifugation. Discard the plasma and platelet-containing upper layer and use a sterile pipette to transfer the mononuclear cell container buffy coat into a new sterile 50 milliliter tube. Fill the tube with up to 50 milliliters of PBS for washing of the mononuclear cells by centrifugation.Then, resuspend the pellet in 10 milliliters of fresh PBS for counting and resuspend the cells at a 1x10^7 cells per milliliter concentration in fresh PBS on ice. To stain the cells for magnetic bead-binding, first transfer 1-2x10^6 cell aliquots into five milliliter polystyrene tubes for each fluorescence compensation and Fluorescence Minus One control as appropriate. Next, collect the remainder of the cell sample by centrifugation and resuspend the pellet to a 3-6x10^7 cells per milliliter concentration in sterile filtered cold FACS buffer.Split the cell solution equally into four FACS tubes on ice and add human Fcgamma receptor blocking reagant to these four experimental sample fractions. At the end of the nonspecific-binding blocking incubation, add the appropriate fluorescence conjugated cell-surface staining antibodies to the cells in all four sample fractions. Then, add the appropriate biotinylated antigen to fractions A and D followed by the addition of a sufficient volume of unlabeled antigen to fraction B such that the majority of the receptors with an affinity of at least 1x10^6 molar are blocked.Incubate all of the samples for 30 minutes on ice followed by a 30 minute incubation of fraction B with the appropriate biotinylated antigen. At the end of the incubations, wash the cells two times in one milliliter of ice cold FACS buffer per 5x10^7 cells. And resuspend the pellets in one milliliter of 2%formaldehyde per 5x10^7 cells for five minutes in the dark on ice.After fixation, wash cells two more times as just demonstrated. And resuspend the pellets in one milliliter of fresh ice cold FACS buffer per 5x10^7 cells. Then label the cells with one to two micrograms of the appropriate secondary Streptavidin Conjugated Antibody for 20 minutes in the dark on ice and wash the cells two more times.For magnetic nanoparticle-based enrichment, resuspend the A, B, and C cell fractions in one milliliter of cold Separation Buffer per 5x10^7 cells and filter the cells through individual 40 microliter strainers to eliminate any clumps. Next, add the fluorescent dye nanoparticles at the appropriate concentration for 10 minutes in the dark at four degrees Celsius with rotation and wash the samples two times in one milliliter of cold Separation Buffer. Resuspend the pellets in one milliliter of cold Separation Buffer per 5x10^7 cells.Then equilibrate three magnetic columns on their appropriately-sized magnets with three milliliters of Separation Buffer each, allowing the entire volume of Separation Buffer to run through each column into a waste container. Now place one labeled 15 milliliter conical tube under each column and add the magnetic particle labeled cells to their respective columns. When the entire volume of each cell fraction has passed through each column, add three milliliters of fresh ice cold Separation Buffer to the top of each column, collecting the effluents in the labeled tubes.After the second wash has completely drained through the columns, place the tubes of negatively selected cells from each fraction on ice and transfer the columns into new 15 milliliter tubes for each fraction. Fill each column with approximately six milliliters of fresh Separation Buffer and immediately plunge the buffer through the columns to collect the magnetically-bound cells. Then collect both the positive and negative cell fractions by centrifugation and resuspend the pellets in the appropriate solution for the planned downstream analysis.In these graphs, the frequency of tetanus-toxoid reactive B cells from an individual who was last vaccinated against tetanus more than a decade ago demonstrates the ability of this method to be used to enrich the tetanus-toxoid-binding cells approximately seven-fold with the purity of about 4%The addition of a 50-fold excess of unlabeled tetanus toxoid to the cells from a subject who was vaccinated about three years prior to the blood draw. Before biotinylated antigen is added, the tetanus-toxoid binding is decreased by 83%demonstrating the specificity of antigen binding. When the biotinylated antigen is omitted from the procedure, only 0.2%of the B cells still bind, presumably, reflecting the binding of some non-tetanus antigen in the absorbent.These graphs demonstrate how the frequency of tetanus-toxoid-specific naive B cells from a blood sample drawn seven days after the booster vaccination of a healthy subject, decreases from 84%to 64%after the boost. While the percentage of memory and plasmablast populations increases from 16%to 36%and zero to 40%respectively. Further, representative ELISPOT data from a peripheral blood sample obtained seven days post-boost from a healthy subject illustrate the four-fold enrichment in tetanus-toxoid antibody-secreting cell frequency observed in the isolated tetanus-toxoid-binding B cells.Once mastered, this technique can be completed in about three to four hours with all the proper controls. The method can be combined with secondary assays, for example, transfer of cells into adopted recipients for subsequent studies of cell fate and function. After watching this video, you should have a good understanding of how to isolate and enrich for rare antigen-binding B cells from human peripheral blood using the proper controls.

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Research

JoVE Journal

Immunology and Infection

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.  ...

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 ...

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 ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. However, the literature reports contrasting data on the prevalence of myocardial B-cells. B-cells have been reported to be both among the most prevalent immune cells in the rodent heart or to be present, but at a markedly lower prevalence than myeloid cells, or to be quite rare. Similarly, several groups have described that the number of myocardial B-cells increases after acute ischemic myocardial injury, but one group reported no changes in the number of B-cells of the injured myocardium. Implementation of a shared, reproducible method to assess the prevalence of myocardial B-cells is critical to harmonize observations from different research groups and thus promote the advancement of the study of B-cell myocardial interactions. Based on our experience, the seemingly contrasting observations reported in the literature likely stem from the fact that murine myocardial B-cells are mostly intravascular and connected to the microvascular endothelium. Therefore, the number of B-cells recovered from a murine heart is exquisitely sensitive to the perfusion conditions used to clean the organ and to the method of digestion used. Here we report an optimized protocol that accounts for these two critical variables in a specific way. This protocol empowers reproducible, flow cytometry-based analysis of the number of murine myocardial B-cells and allows researchers to distinguish extravascular vs. intravascular myocardial B-cells.

Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...