Abstract
The memory B-cell (MBC) ELISpot assay is the main technique used to measure antigen-specific MBCs as a readout of humoral immune memory. This assay relies on the ability of MBCs to differentiate into antibody-secreting cells (ASC) upon polyclonal stimulation. The total number of IgG+ ASCs generated by mitogen-stimulation is often used as a reference point; alternatively antigen-specific MBCs are expressed as a frequency of post-culture peripheral blood mononuclear cells (PBMC) as a surrogate for absolute frequencies. Therefore, it is important to know whether IgG+ B-cells are uniformly expanded during the preceding mitogen-culture as a true reflection of MBC frequencies ex vivo. We systematically compared B-cell phenotype and proportions before and after mitogen stimulation in cultures of 269 peripheral blood mononuclear cell samples from 62 volunteers by flow cytometry and analyzed the number of resulting ASCs. Our data show that the number of total IgG+ ASCs detected by ELISpot after mitogen stimulation correlates with the proportion of IgG+ MBCs ex vivo, highlighting its general robustness for comparisons of study cohorts at group level. The expansion of total and IgG+ B-cells during mitogen-stimulation, however, was not identical in all cultures, but influenced by size and composition of the ex vivo B-cell compartment. The uncorrected readout of antigen-specific MBCs per million post-culture PBMCs therefore only preserves the quality, but not the magnitude of differences in the ex vivo MBC response between groups or time points, particularly when comparing samples where the B-cell compartment substantially differs between cohorts or over time. Therefore, expressing antigen-specific cells per total IgG+ ASCs is currently the best measure to correct for mitogen-culture effects. Additionally, baseline information on the size and composition of the ex vivo B-cell compartment should be supplied to additionally inform about differences or changes in the size and composition of the ex vivo MBC compartment.
Highlights
Humoral immunity is crucial to combat many infections and to provide protection against re-infection and after vaccination
With the exception of a slight dip in total B-cells four weeks after the last immunization, both the size of the total and IgG+ circulating B-cell compartment, and the proportions of the different memory B-cell (MBC) subsets remained constant over time in the 53 donors from which multiple samples were available (Table S1 and Table S2), highlighting that the interventions the volunteers underwent in the clinical trials had no biasing effect on the samples analyzed
When comparing the number of IgG+ B-cells determined by flow cytometry to the number of IgG+ ACSs detected by ELISpot, both per million peripheral blood mononuclear cells (PBMC) post-culture, we found that the number of IgG+ B-cells, in a similar range, was slightly but significantly higher than that of IgG+ antibody-secreting cells (ASC) (Figure 3A; p = 0.003)
Summary
Humoral immunity is crucial to combat many infections and to provide protection against re-infection and after vaccination. MBC-secreted antibodies can be quantified (by Enzyme-linked ImmunoSpot assay (ELISpot) or ELISA) following a pre-activation step using mitogens to differentiate MBCs into ASCs [5]. This method is readily applicable to large numbers of samples and antigens (provided sufficient cells are available from each sample), without the need for fluorescent labelling, which can be challenging for individual antigens [6]. This is done to take into account inter-individual variations in total MBC frequencies when comparing across age groups [11,13] that differ not just in their antigen-experience and the size and composition of the (memory) B-cell compartment [11,14] This readout, does not correct for expansion and potential skewing of the MBC compartment during mitogen culture. None of these studies investigated the effect of mitogen culture on B-cell expansion, which would have affected both post-culture read-outs
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