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

Summary

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

Abstract

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

Introduction

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 sorting^1,2,3,4,5,6. 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' disease, and Hashimoto'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 fluorochrome^8,9,10,11. 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 small^12,13,14. 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 naïve, 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.

Protocol

1. Isolation of Human PBMCs

1. 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.
2. Mix whole blood 1:1 with sterile, room temperature magnesium and calcium free phosphate-buffered saline (PBS).
3. 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 °C, but warm to room temperature before use.
4. 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.
5. Centrifuge at 400 x g for 30 min at 20 °C with the brake turned off.
6. Remove the upper layer, which contains plasma and platelets, being careful not to disturb the mononuclear cell layer below.
7. Using a sterile pipette, collect the mononuclear cell layer (buffy coat) and place into a separate sterile 50 ml conical tube.
8. Add PBS to the cell suspension to a volume of 50 ml. Centrifuge at 400 x g for 10 min at 20 °C with brake.
9. Remove supernatant and resuspend pelleted cells in 10 ml of PBS. Count cells and determine viability using a hemocytometer or automated cell counter.
10. Resuspend cells at 10⁷ cells/ml and place on ice until ready for further processing.

2. Staining of Cells

1. 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 °C with brake.
2. 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.
   1. 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.
3. Add human FcγR blocking reagent to fractions A-D on ice to prevent binding of antibodies to Fc receptors.
   NOTE: Follow manufacturer's instructions for appropriate conditions.
4. 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.
5. 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 µM). 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.
6. 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 µM.
   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).
7. 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.
8. 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 °C.
9. 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 – for downstream analysis, such as in ELISPOTs or adoptive transfers, omit the fixation step.
10. 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 °C. Resuspend in 1 ml cold FACS buffer per 5 x 10⁷ cells.
11. Add 1-2 µg streptavidin-far-red-fluorescent dye/ml and incubate on ice for 20 min in the dark. Titrate the amount of streptavidin for each application.
12. 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 °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.

3. Magnetic Nanoparticle-based Enrichment

1. 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 µm filter to eliminate any clumps that could clog the column.
2. 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 µl suspension per 5 x 10⁷ cells/ml. Rotate in the dark at 4 °C for 10 min.
3. Wash twice with 1 ml cold separation buffer at 400 x g for 5 min at 4 °C. Resuspend cells in 1 ml of cold separation buffer per 5 x 10⁷ cells.

4. Magnetic Enrichment Using LS or LD Columns

1. 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.
2. Place three 15 ml conical tubes labeled "Fraction A/(B)/(C) negative", 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.
3. 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.
4. Remove the column from the magnetic field and place on top of a 15 ml conical tube labeled "Fraction A/(B)/(C) positive", for positively selected cells.
5. 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.
6. To increase purity, the positively selected cells can be further enriched using a second clean column, repeating the procedure.
7. Spin both the negatively and positively selected fractions at 400 x g for 10 min at 4 °C and suspend in desired final volume. Proceed with downstream analysis, such as FACS, ELISPOT or adoptive transfer.

Results

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 ...

Discussion

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antigen of interest	variable	variable	At least 100 μg to biotinylate easily; if using protein try to use protein that has been validated by ELISA. It must be carrier  protein free.
Biotin for labeling; e.g. EZ link Sulfo-NHS-LC-Biotin	Thermo Scientific	21335	Biotin is available in different formulations, such as those containing  various length spacers, so the type used should be determined by the researcher
Streptavidin-Alexa Fluor 647	Invitrogen	S21374	Can obtain from other suppliers.
Anti-Cy5/Anti-Alexa Fluor 647 Microbeads	Miltenyi Biotech	130-091-395
LS Columns	Miltenyi Biotech	130-042-401
MACS manual separators	Miltenyi Biotech	variable
Formaldehyde			Dilute to 2% with PBS; optional if downstream assay requires live cells
PBS without calcium and magnesium
Ficoll-Paque PLUS	GE Healthcare	17-1440-02
Whole blood in heparinized collection tubes
FACS buffer (PBS + 1% BSA + 0.01% sodium azide)
Separation buffer (PBS + 0.5% BSA + 2 mM EDTA)
50 ml conical tubes
15 ml conical tubes
1.5 ml Eppendorf tubes
Surface marker reactive antibodies, Fc Block, live/dead discriminating stain, if needed
ELISPOT supplies, if needed