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,....

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 tub....

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