Enumeration of antigen-specific MBC is critical for in-depth analysis of humoral immune responses following vaccination or infection, but remains challenging due to their relatively low frequencies. In this study, we describe the development of two independent and cross-validated methods for the quantitation of antigen-specific MBC in human clinical samples. The FC approach provides the simultaneous detection of TT- and DT-specific MBC by direct antigen-binding, while the ELISA-based LDA quantitates MBC on the basis of their functional ability to proliferate and differentiate into antibody secreting cells and allows for enumeration of up to 10 antigen-specific MBC populations from a single experiment.
FC offers the ability to directly examine antigen-specific MBC, and has been commonly used to investigate MBC responses in mice (Hayakawa et al., 1987
; McHeyzer-Williams et al., 1993
). However, frequencies for antigen-specific MBC populations in these animal models can be relatively high, comprising up to ~1% of total splenocytes in certain instances (McHeyzer-Williams et al., 1993
). This is in contrast to human clinical samples in which MBC frequencies may be as low as ~0.003% of total B cells for specific populations (Leyendeckers et al., 1999
). With such low frequencies, even a limited number of false-positive events can profoundly hinder analysis (Townsend et al., 2001
). To increase the specificity of direct MBC staining by FC, a “dump channel” is often used to gate out non-specific, antigen-binding cells (McHeyzer-Williams and McHeyzer-Williams, 2004
). In our studies, we avoided extensive negative gating by employing a dual staining approach, similar to that described in a transgenic B cell receptor mouse model (Townsend et al., 2001
) and found that it dramatically improved specificity without reducing the sensitivity of MBC quantitation (). This approach was further optimized by the addition of stringent antigen-mismatch staining controls to confirm antigen specificity (). This antigen-mismatch approach, wherein samples are divided into two fractions and stained with dual antigen-specific reagents (e.g. TT-FITC + TT-APC), or a single antigen-specific reagent in addition to an irrelevant “mismatch” fluorescently labeled protein reagent (e.g. TT-FITC + HSA-APC) not only provides an important specificity control but also facilitates optimal placement of the region gate used for MBC quantitation. This stringent internal specificity control is particularly important for humans and NHP, in which genetically matched, naive/unvaccinated individuals are often not available for comparison.
The dual staining FC approach works well for simple protein antigens such as TT and DT, but staining B cells with highly repetitive and complex multi-protein antigens has previously proven difficult to perform due to substantial non-specific binding (Doucett et al., 2005
). To expand the range of antigen-specific MBC populations that can be measured in clinical samples, and to cross-validate the FC approach that we used to measure MBC frequencies specific to single protein antigens, we developed an optimized and highly sensitive ELISA-based LDA for determining MBC numbers. Purified CD20+
B cells were stimulated polyclonally in vitro
under limiting dilution conditions and supernatants from the limiting dilution cultures were tested by ELISA for reactivity against several antigens. One advantage of this LDA over other previously described methods is that the MBC frequencies can be quantitated on the basis of the total B cell population rather than as a proportion of B cell subpopulations capable of antibody production following polyclonal activation (Crotty et al., 2004
). Another advantage of this LDA over other LDA methods that use antigen-specific stimulation for MBC quantitation (Slifka and Ahmed, 1996
) is that polyclonal stimulation results in antibody production by all potential MBC specificities (instead of antibody responses elicited against only a single antigen) and therefore cell culture supernatants can be used to measure MBC responses specific for virtually any antigen that works well for ELISA quantitation of serum antibody responses. The LDA is a functional assay designed to measure MBC capable of dividing and differentiating into antibody secreting cells and therefore provides different biological information about MBC than that obtained by the FC approach, which only measures antigen-specific binding and not function. On the other hand, the FC approach facilitates clear phenotypic characterization of the antigen-specific MBC populations since multiple internal or cell surface markers can be directly visualized on the antigen-binding cells.
In , approximately 1/23 CD20+
B cells (containing a mixture of naive and antigen-experienced B cells) were identified by this optimized LDA. This was typical of 13 clinical samples tested (average=1/40, range=1/15 to 1/84). However, CD20+
B cells comprised just 15.7±5.9% (range = 8.4% to 24.6%) of peripheral B cells in subjects tested (n
= 13, data not shown) and we reasoned that the low cloning efficiency observed in these experiments was due to naive B cells (~84% of peripheral B cells) being largely incapable of differentiation into IgG-secreting cells under stimulation conditions employed here. The bulk of MBC reside in the class-switched, IgD−
B cell population (Hayakawa et al., 1987
) and to determine if naive (IgD+
) B cells responded differently to polyclonal stimulation than Ig-class switched (IgD−
), we magnetically sorted PBMC into IgD+
fractions for a subgroup of individuals (n=4) and performed the LDA. For the IgD+
B cells, total Ig-producing precursor frequencies averaged 1/21 (range=1/7 to 1/33), while IgG-producing precursor frequencies averaged only 1/844 (range=1/154 to 1/2030). In contrast, an average of 1/3.7 (range=1/2.5 to 1/5.8) IgD−
B cells produced antibody after in vitro
polyclonal stimulation under limiting dilution conditions (data not shown). This may still represent an underestimate of MBC activation under LDA conditions since a proportion of the gated CD20+
B cells may be circulating marginal zone B cells displaying an IgDlo
(as well as IgM+
) phenotype (Weller et al., 2004
) and not necessarily represent antigen-induced MBC. Using these LDA conditions to enumerate TT-specific MBC from clinical samples, we found a significant correlation with the frequencies determined by FC (p = 0.0002, ). As might be expected when relying on a functional readout, the LDA was somewhat less sensitive than FC, detecting ~70% of the response observed through direct FC staining. However, in contrast to the FC approach, which was limited to analysis of two simple protein antigens, the LDA described here was able to quantitate antigen-specific MBC frequencies for up to 10 different antigens, including complex viral antigens such as vaccinia, measles, and mumps ( and data not shown). This method will be useful for determining MBC frequencies for any antigen that can currently be studied by ELISA, greatly expanding the utility of this approach for measuring functional MBC responses to multiple antigens.
The mechanisms which maintain MBC, as well as the respective roles of MBC and plasma cells in sustaining long-term antibody responses in humans, remain controversial (Slifka, 2004
; Manz et al., 2005
; McHeyzer-Williams and McHeyzer-Williams, 2005
; Amanna et al., 2006
) making the quantitative analysis of antigen-specific MBC a timely and relevant topic. The assays presented here allow for the enumeration of MBC with different antigen-specificities and will aid in addressing these fundamental questions of immunological memory.