Research paperHigh efficiency human memory B cell assay and its application to studying Plasmodium falciparum-specific memory B cells in natural infections
Introduction
A hallmark of adaptive immunity is Ag-specific immunological memory, the ability to respond more rapidly and robustly to re-exposure to an Ag. Indeed, all current vaccines are predicated on the phenomenon of immunological memory (Sallusto et al., 2010). However, despite its importance we still have an incomplete understanding of the cellular and molecular mechanisms that underlie the generation, maintenance and activation of immunological memory. For many infectious diseases neutralizing antibodies (Abs) play a critical role in protective immune responses (Sallusto et al., 2010), and thus the mechanisms that underlie the generation and maintenance of B cell memory are of considerable interest. Of particular interest is an understanding of the acquisition of B cell memory in infectious diseases for which we currently have no vaccines, including malaria.
Long-term humoral memory is encoded in both memory B cells (MBCs) and long-lived plasma cells (LLPCs) that are generated during a primary immune response to vaccination or infection. LLPCs reside in the bone marrow and are responsible for maintaining serum antibody (Ab) levels in the absence of Ag re-exposure (Radbruch et al., 2006, Wrammert et al., 2009). Because of their inaccessibility to routine sampling in humans, LLPCs are rarely directly assayed. However, the presence of Ag-specific Ab in serum in the absence of re-exposure to an Ag is likely an accurate reflection of the presence of Ag-specific LLPCs. MBCs are responsible for the rapid, high titer, high affinity secondary Ab responses elicited upon re-exposure to a pathogen or booster vaccination (Radbruch et al., 2006, Wrammert et al., 2009). MBCs reside both in the lymphoid tissues and in the peripheral circulation. The results of a recent study in humans receiving a smallpox vaccine provided evidence that although the majority of vaccine-specific MBCs were in the spleen, the frequency of Ag-specific MBCs in the spleen paralleled the frequency observed in peripheral blood throughout the course of the response (Blink et al., 2005). Furthermore, a recent study using a mouse model of malaria indicated MBCs and PCs in peripheral blood mononuclear cells (PBMCs) reflect Plasmodium-specific B cell responses in the spleen and bone marrow (Nduati et al., 2010). Based on these observations, the frequencies of MBCs in peripheral blood appear to be a good indication of the total number of MBCs in an individual.
Here we focus on the development of B cell memory to Pf malaria because it kills nearly one million people annually and there is no vaccine. Immunity to clinical malaria is slow to develop requiring years of repeated infections (Langhorne et al., 2008, Crompton et al., 2010, Weiss et al., 2010). Abs are known to play a central role in protection from malaria (Cohen et al., 1961) but the B cell biology that underlies the slow acquisition of immunity is just beginning to be investigated. Therefore it is important to develop highly efficient assays that provide the tools for assessing MBCs in the PBMCs of individuals acquiring malaria immunity through natural infection. Evidence is accumulating that the immune systems of individuals living in malaria endemic areas may differ from those in non-malaria endemic areas. For example, in one study we determined that individuals in Mali, in an area of high malaria transmission, have greatly expanded populations of atypical or ‘exhausted’ MBCs (Weiss et al., 2009). In another, we found that Malian adults were relatively refractory to the potentiating effects of CpG-containing vaccines as compared to individuals in the U.S. in that while CpG-containing vaccines resulted in a significant increase in the number of MBCs and the Ab titer in the U.S., there was no difference in MBC number or antibody titer when CpG-containing or non-CpG-containing vaccines were administered to Malian adults (Crompton et al., 2009). Thus, it is essential that assays to detect Pf-specific MBCs are validated in field samples from individuals living in malaria endemic areas.
An assay frequently used to detect Ag-specific human MBCs, described by Crotty et al., 2004 (Crotty et al., 2004), relies on the selective ability of MBCs to proliferate and differentiate into Ab secreting cells (ASCs) in vitro in response to a combination of pokeweed mitogen (PWM), fixed S. aureus, Cowan strain (SAC) and the TLR9 agonist CpG oligonucleotide (ODN-2006) over a five to six day culture period. Ag-specific and total ASCs were quantified in ELISPOT assays using plates coated with either Ag or human Ig-specific antibodies to capture Ag-specific and total IgG, respectively. Crotty et al. showed that peripheral blood MBCs (defined as CD19+CD20+CD27+) from individuals immunized with anthrax vaccine differentiated into anthrax protective antigen (PA)-specific ASCs in this assay, but naïve B cells (defined as CD19+CD20+CD27−) did not. PA-specific MBCs represented up to 2% of circulating IgG+ B cells in immune individuals and were essentially undetectable in non-immune individuals. Thus, this assay provided a means of identifying Ag-specific MBCs in human peripheral blood.
Using peripheral blood samples from vaccinated U.S. individuals we determined the efficiency of this assay by enumerating the actual number of tetanus toxoid (TT)-specific MBCs in a given sample using a sensitive and specific flow cytometry technique (Amanna and Slifka, 2006) and comparing this number to the number of MBCs which respond to the stimulation cocktail used by Crotty et al. We report that only ~ 10% of TT-specific MBCs enumerated by flow cytometry respond in a limiting dilution version of the in vitro assay described by Crotty et al. and that the efficiency of this assay could be significantly improved, to nearly 100%, by the addition of IL-10 to the five day culture. This increase in efficiency in a method that can be done with whole PBMCs and does not require isolation of B cells as do other highly efficient assays (Amanna and Slifka, 2006) makes this protocol valuable in field studies in malaria endemic areas, where isolation of B cells is impractical due to both sample number and sample size. We provide evidence that this modified assay can be successfully used to quantify Pf-specific MBCs in semi-immune adults in Kenya. The increased efficiency of this modified assay allows for the measurement of MBCs from smaller blood volumes than was possible with the original assay that should facilitate field studies, particularly those involving children.
Section snippets
Samples and sample preparation
Peripheral blood was obtained from healthy, anonymous, adults at the National Institutes of Health blood bank and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood or elutriated mononuclear cell buffy coats obtained by lymphapheresis within 6 h of blood collection, using by density gradient centrifugation. Samples were diluted in an equal volume of PBS, layered over a 20% volume of Ficoll–Hypaque (Amersham Biosciences) and centrifuged for 20 min at room temperature at 1800 ×
Quantifying antigen-specific MBCs by flow cytometry
To quantify Ag-specific MBCs in vaccinated U.S. individuals we used a sensitive flow cytometry-based assay described for detecting TT-specific MBCs by Amanna and Slifka (Amanna and Slifka, 2006). We chose to measure TT for the assessment of the MBC ELISPOT assay because our attempts to detect Pf-specific MBCs using the Ags apical membrane antigen 1 (AMA1) and merozoite surface protein 1 (MSP1), were unsuccessful, because of the instability and non-specific binding of these proteins. PBMCs were
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