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

Summary

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.

Abstract

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 naïve 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.

Introduction

B cells play a central role in the development of humoral immunity. They initially develop in the bone marrow and enter the blood stream as naïve B cells, which can migrate into the lymphoid tissues, such as the spleen, lymph nodes, and tonsils, for further development. Upon Ag encounter, some naïve B cells migrate into lymphoid follicles, where germinal center B cells can differentiate into memory B cells and plasmablasts (PBs)/plasma cells (PCs). While most PBs/PCs egress into the blood stream, a few eventually reside in the bone marrow to undergo terminal differentiation into long-lived PCs¹. B cells in circulation are heterogeneous, and at ste....

Protocol

Human peripheral blood must be obtained from healthy donors under informed consent, and the use of blood samples must conform to the approved guidelines established by individual institutional review boards. In this study, the protocol to use human blood in a demonstration of the results of flow cytometry (Figure 1) and ELISpot assays (Figure 3) was approved by the Internal Review Board of National Taiwan University Hospital (protocol number 201307019RINB).

1. Isolation and Purification of Human Peripheral Blood B Cells

1. Draw ~ 10 mL of blood from the median cubital vein (in the cubital fossa anterior to the elbow) i....

Results

PBMCs were depleted of RBCs and adherent cells (steps 1.2 to 1.7). An aliquot (2 x 10⁶) of cells were subjected to a flow cytometric analysis to illustrate the populations of naïve B cells, memory B cells, and PBs/PCs in peripheral blood (Figure 1). In this donor's PBMCs, about 10% of the lymphocytes were CD19⁺ B cells. In the B-cell compartment, the percentage of CD19⁺CD27^- naïve B cells was around 50%. On the other hand, about.......

Discussion

Isolation and Purification of Human Peripheral Blood B Cells

Normally, RBCs can be efficiently ruptured and cleared by lysis buffer (step 1.2). It is important not to incubate PBMCs with the RBC lysis buffer longer than 5 min, as cell viability might be affected by the ammonium chloride. Alternatively, RBCs and platelets can be simultaneously removed by the following protocol.

Mix fresh whole blood with acid-citrate-dextrose (ACD) buffer (39 mM citric acid, 75 mM sodiu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD Vacutainer K2E	BD Biosciences	367525	10 mL tube
Ficoll-Paque Plus	GE Healthcare	17-1440-02	endotoxin-free
Trypan blue 0.5% solution	Biological Industries	03-102-1B
IMag Human B lymphocyte enrichment set	BD Biosciences	558007
Biotinylated CD27 mAb	Biolegend	302804	clone O323
Streptavidin magnetic microbeads	BD Biosciences	9000810
15 mL Falcon tubes	BD Falcon	352196
Blue nylon mesh cell strainer, 40 μm	BD Falcon	352340
Anti-human CD19-APC	Biolegend	302212	clone HIB19
Anti-human CD27-eFluor 450	eBioscience	48-0279-42	clone O323
Anti-human CD38-PE-Cy7	Biolegend	303516	clone HIT2
Anti-human CD38-PE-Cy7	BD Biosciences	560677	clone HIT2
Anti-human CD45-FITC	Biolegend	304006	clone HI30
Anti-human CD45-FITC	BD Biosciences	555482	clone HI30
Anti-mouse/rat/human CD27-PerCP Cy5.5	Biolegend	124213	clone LG.3A10
Anti-human CD27-PerCP Cy5.5	BD Biosciences	65429	clone L128
Anti-human CD19-FITC	Miltenyi Biotec	130-098-064	clone LT19
Anti-human CD19-FITC	GeneTex	GTX75599	clone LT19
Anti-human CD20-FITC	BD Biosciences	555622	clone 2H7
biotinylated anti-human CD27	Biolegend	302804	clone O323
biotinylated anti-human CD27	eBioscience	13-0279-80	clone O323
7-aminoactinomycin D (7-AAD)	BD Biosciences	559925
CpG (ODN 2006)	InvivoGen	tlrl-2006	type B CpG
Recombinant human IL-2	PeproTech	200-02
Recombinant human IL-10	PeproTech	200-10
Recombinant human IL-21	PeproTech	200-21
Recombinant human sCD40L	PeproTech	310-02
Protein A of S. aureus Cowan (SAC)	Sigma-Aldrich	82526
Pokeweed mitogen (PWM)	Sigma-Aldrich	L9379
MultiScreen filter plates, 0.45 µm pore size	Merck Millipore	MSIPS4510	sterile, clear 96-well filter plate with hydrophobic PVDF membrane
BCIP/NBT solution	Sigma-Aldrich	B6404
BCIP/NBT single reagent, alkaline phosphatase substrate	Merck Millipore	ES006
Human IgG	Jackson ImmunoResearch	009-000-003
Human IgG, Fc fragment	Jackson ImmunoResearch	009-000-008
F(ab')2 fragment of goat anti-human Ig (IgG + IgM + IgA)	Jackson ImmunoResearch	109-006-127
Goat anti-human IgG-alkaline phosphatase, Fcγ fragment specific	Jackson ImmunoResearch	109-055-008
Goat anti-human IgM-alkaline phosphatase, Fcµ fragment specific	Jackson ImmunoResearch	109-055-043
Goat anti-human IgG-peroxidase, Fcγ fragment specific	Jackson ImmunoResearch	109-035-008
Goat anti-human IgM-peroxidase, Fcµ fragment specific	Jackson ImmunoResearch	109-035-095
BD ELISPOT AEC substrate kit	BD Biosciences	551951
C.T.L. ImmunoSpot analyzer	C.T.L.