CD27⁺ switched memory cells (IgG⁺/IgA⁺/IgE⁺)

Memory B cells in human have been originally described as IgD⁻CD27⁺ cells, which have undergone the CSR and SHM process, in comparison to IgM⁺IgD⁺CD27⁻ naïve B cells. These “classical” memory cells express IgG, IgA, or IgE and they derive from T cell-dependent (TD) response in the GC (Pascual et al., 1994). As discussed below, IgM⁺CD27⁺ cells are also memory subsets, but may have roles differing from the class-switched populations. The general function of these cells is to induce a rapid and robust response after re-exposure to a pathogen or antigen by the differentiation to high affinity plasma cells (Arpin et al., 1995). These cells recirculate in the peripheral blood, spleen, and tissues to survey for their cognate antigen (Klein et al., 1998b; Tangye et al., 1998; Montoya et al., 1999). It is interesting to consider that the various secondary, class-switched isotypes of Ig may represent a population multiplier. Clearly, the various isotypes utilize different intracellular domains as BCRs and are associated with specific functional roles such that an IgA “mucosal memory” cell may be quite distinct functionally from IgG1 or IgG4 expressing memory cells.

Tissue-specific and “tissue-like” memory: CD27⁻FCRL4⁺ memory cells

The advent of CD27 as a general memory marker allowed great strides in the study of the memory compartments of human B cells. It came as a great surprise when B cell subsets that appeared to be newly discovered members of the memory compartment were located but lacked the expression of CD27.

In 2003, work was being done to characterize a new set of B cell inhibitory receptor genes, known as the Ig superfamily receptor translocation associated genes (IRTA; Miller et al., 2002; Falini et al., 2003). The gene IRTA1, encoding the orphan receptor Fc-receptor-like-4 (FCRL4) molecule, was present on mucosally associated B cells and contained several immunoreceptor tyrosine-based inhibitory motifs (ITIMs); these were later shown to be potent negative regulators of BCR signaling (Miller et al., 2002; Ehrhardt et al., 2003; Falini et al., 2003). The inhibitory function of FCRL4 in B cells was seen to be mediated by the protein SHP-1 (Ehrhardt et al., 2003).

When this population was later located in the tonsillar tissue of humans, it was found that the cells did not express the memory marker CD27 (Ehrhardt et al., 2005). This B cell population was not seen in the blood, spleen, or bone marrow of human patients (Miller et al., 2002; Ehrhardt et al., 2005). However, these tissue localized B cells did have evidence of SHM, CSR to IgG and IgA, and lacked expression of GC markers (Ehrhardt et al., 2005). These cells also carried conventional markers of activation as well as higher CD20 expression (Ehrhardt et al., 2005). Interestingly, these CD27⁻FCRL4⁺ B cells were larger than their CD27⁺FCRL4⁻ counterparts and could be stimulated to secrete Ig using T cell cytokines but not BCR stimulation (Ehrhardt et al., 2005). A gene array analysis of FCRL4⁺ versus FCRL4⁻ cells from tonsillar tissues has revealed that these cells differ in their transcriptome profiles. Expression of the transcription factors RUNX1 and RUNX2 appears to be a major difference between these populations (Ehrhardt et al., 2008). Moreover, the FCRL4⁺ memory B cells have a distinct expression patterns for receptors, signal transduction molecules, and transcription factors (Ehrhardt et al., 2008). The function of these cells is not defined but they may be important in the mucosal immune response. The discovery of a new subset of tissue-specific memory B cells that were not identifiable by CD27 staining spurred the notion that the B cell memory compartment was much more intricate than initially assumed.

Recent work done in the surveying of B cell responses to HIV has revealed a population of B cells in the peripheral blood that appeared to share a similar phenotype to tissue-specific CD27⁻FCRL4⁺ B cells described in tonsillar tissues (Ehrhardt et al., 2005; Moir et al., 2008). Interestingly, these HIV-infected viremic patients were seen to have this CD27⁻FCRL4⁺ population present in their peripheral blood at a frequency of 19% of total B cells, which is in stark contrast to the <4% of the same population in healthy individuals (Moir et al., 2008). This cell population, deemed “tissue-like memory B cells,” expressed markers of past SHM, albeit at a lower frequency and diversity than normal CD27⁺ memory cells, and showed a reduced tendency to proliferate in response to cognate antigen (Moir et al., 2008). These tissue-like memory B cells also expressed not only FCRL4, but a number of other inhibitory receptors and chemokine receptors that would reduce the likelihood of B–T cell interaction (Moir et al., 2008). Further, these tissue-like memory B cells had reduced capability to produce ASCs but were the cell population most specific for the HIV gp120 protein (Moir et al., 2008). Specific siRNA knockdown of FCRL4 and other inhibitory receptors led to a rescue of Ig secretion and proliferation in these tissue-like memory B cells (Kardava et al., 2011). This phenotype resembled what had been seen in exhausted CD8⁺ T cells (Day et al., 2006), giving credence to the idea that this was an “exhausted” memory B cell population (Moir et al., 2008).

Since the identification of an exhausted population of B cells, similar phenotypes have been witnessed in other chronic diseases such as malaria and hepatitis-C (Weiss et al., 2009; Charles et al., 2011). It would appear that chronic infections have means to alter human B cell populations to their own benefit; overstimulation of B cells can be quite deleterious to the host and needs to be properly regulated. Vaccines that safely overcome this exhaustion phenotype may be necessary in order to develop proper immunity to these chronic, dangerous diseases.

CD27⁺ IgD⁺IgM⁻ memory cells

Naïve B cells typically express both IgM and IgD isotypes on their surface, the differential production of these two antibody isotypes is controlled by alternative splicing of the immunoglobulin heavy chain RNA transcripts (Early et al., 1980; Moore et al., 1981; Kinashi et al., 1987). Interestingly, there is a population of antigen experienced human CD27⁺IgD⁺IgM⁻, “IgD only,” memory B cells in the blood and a IgD⁺IgM⁻ plasma cell population that reside specifically in respiratory mucosal linings of humans (Liu et al., 1996; Arpin et al., 1998; Klein et al., 1998a).

These cells were initially discovered in 1989 during investigations of human myeloma patients (Yasui et al., 1989; White et al., 1990). These IgD⁺IgM⁻CD27⁺ memory B cells or “Cδ class-switched” cells are the product of a class-switch event at the Cμ and cryptic Cδ switch recombination regions of the Ig heavy chain or, interestingly, by a separate non-classical homologous recombination event (Yasui et al., 1989; White et al., 1990). IgD⁺IgM⁻ B cells carry some of the largest SHM burdens seen to date in their Ig genes; along with having a large skewing for use of lambda light chains (Arpin et al., 1998; Klein et al., 1998a; Zheng et al., 2004) as well as highly restricted VH gene repertoires (Wilson et al., 2000; Zheng et al., 2004; Koelsch et al., 2007; Seifert et al., 2009), with 50–60% of the BCRs being encoded by one of only two heavy chains; VH3–30 or VH4–34. VH4–34 has long been associated with B cell autoimmunity (Pascual et al., 1991, 1992; Pugh-Bernard et al., 2001). This plus high levels of receptor editing for both the heavy and light chain loci, combined with higher auto- and poly-reactivity scoring in healthy individuals have implicated these cells in being uniquely selected during B cell development and immune responses (Wilson et al., 2000; Zheng et al., 2004; Koelsch et al., 2007). B1 and marginal zone cells in mice also express poly-reactive prone BCRs from a highly restricted BCR repertoire (Kearney, 2005), thus one can postulate that like these populations, IgD⁺IgM⁻CD27⁺ might serve more specialized innate-like or tissue-specific roles.

More recently work has been performed on possible effector functions of IgD⁺IgM⁻ B cells that supports the possibility that these cells are to some degree functional analogs to B1 or MZ cells. Plasmablasts from human samples were seen to develop in the respiratory mucosa and produce IgD that was specific for pathogens of the respiratory tract (Chen et al., 2009). Interestingly, the IgD that was produced was seen to bind strongly to the Fc receptors on the surface of basophils (Chen et al., 2009). This crosslinking of IgD on the surface of basophils lead to the release of B cell-activating factors and antimicrobial compounds (Chen et al., 2009). The authors concluded that these IgD⁺IgM⁻ plasma cells and IgD⁺IgM⁻CD27⁺ memory cells appear to play an important role in respiratory mucosal defense via the activation of basophil populations; providing at least one possible function for this mysterious cell population in vivo (Chen et al., 2009).

CD27⁻IgG⁺ and CD27⁻IgA⁺ memory cells

Further heterogeneity in memory B cell subsets has been reported with CD27⁻IgG⁺ B cells that exhibit classic attributes of memory cells, including SHM and expression of co-stimulatory molecules (Fecteau et al., 2006; Wei et al., 2007). CD27⁻IgG⁺ and CD27⁺IgG⁺ memory B cells differed in the distribution of IgG subclasses, with a predominance of IgG2 expression in CD27⁺IgG⁺, while CD27⁻IgG⁺ expressed preferentially IgG3 (Fecteau et al., 2006). In humans, IgG2 antibodies are mainly reactive to polysaccharides while IgG3 antibodies more often react with proteins; the unique isotype distributions between these two populations further suggests that CD27⁻IgG⁺ B cells could be distinct in their function (Fecteau et al., 2006). Moreover, analysis by Berkowska et al. (2011) has led to the conclusions that CD27⁻IgG⁺ cells are derived from early GC reactions, like IgM-only memory cells described below, whereas CD27⁺IgG⁺ cells are derived from consecutive GC reactions based on the replication history, SHM, and CSR data of these two populations. Interestingly, these CD27⁻IgG⁺ cells are expanded in SLE patients compared with healthy controls (Wei et al., 2007). It is not yet known how these cells effect disease progression.

A minor population of memory cells have been described as CD27⁻IgA⁺. These cells have fewer mutations than CD27⁺IgA⁺ cells and they may originate from local GC-independent responses in the gut, such as IgA⁺ B cells from the lamina propria (Berkowska et al., 2011). The function of this population remains to be studied.

Both CD27⁻IgA⁺ and CD27⁻IgG⁺ populations are increased in patients with HIV chronic infection and an increased IgA production by CD27⁻IgA⁺ cells was observed upon CD40 and TLR9 stimulation in vitro (Cagigi et al., 2009). These CD27⁻ isotype-switched memory cells (IgG⁺ and IgA⁺) can be discriminated from CD27⁻ naïve cells by the absence of ATP-binding cassette B1 transporter (ABCB1) activity (Wirths and Lanzavecchia, 2005). Therefore, CD27^– memory cells, in opposition to CD27⁺ memory cells, are essentially disconnected from any further regulation through CD70, the ligand of CD27, supporting once more their distinct roles in immune responses (Fecteau et al., 2006).

The diversity of memory B cells may reflect the different origins and nature of the encounter antigens; leading to different possible functions of these memory B cells. The real contribution of each subset in the immune response should be the aim of future studies.

Human B1 cell populations

In mice, B cells are divided into B1 cells (B1-a and B1-b) and B2 cells (follicular cells and marginal zone cells). B1 and marginal zone (MZ) B cells are innate-like B cells that secrete natural antibodies rapidly in response to infections or tissue injury. The development of B1 cells and their response patterns against antigen have been recently reviewed by Baumgarth (2011). Differences in responses to pathogens between the two related populations reside mainly in the BCR activation. Indeed, in response to various stimuli (S. pneumonia, certain carbohydrates, LPS, and certain cytokines) both B1-a and B1-b cells respond with secretion of poly-reactive IgM antibodies (IgA is also possible in the gut), a low level of cell proliferation, and no apparent BCR signaling (Nisitani et al., 1995; Ohdan et al., 2000; Martin et al., 2001). In contrast, following repeated exposure to TI antigens, only B1-b cells can expand clonally, driven by BCR signaling, and secrete antigen-specific IgM antibodies as a memory response (Defrance et al., 2011).

The existence of B1 cells in humans is controversial; various studies have provided evidence of populations that appear to be similar to the mouse B1 phenotypes, but B1-a and B1-b populations per se still lack definition (Griffin et al., 2011). However, polysaccharide vaccines (TI antigens), such as Pneumovax, confer long-term humoral protection in adult humans, suggesting possible TI memory in humans (Lesinski and Westerink, 2001). Notably, however, unlike the antibody genes of typical T-independent responses in mice, the Pneumovax vaccine induces B cells with highly mutated variable genes, similar to T cell dependent responses (Smith et al., 2009). However, it is impossible to say by this study alone whether these cells obtained their somatic mutations solely through the interaction with the TI antigen alone or whether the cells that were stimulated arose from an already somatically mutated TD memory population; more work must be done here to definitively resolve this issue. There is still considerable debate concerning the function of IgM⁺ memory B cells as human B1 cells. However, there is evidence that IgM⁺ memory B cells are implicated in response to both TI and TD antigens (Tangye and Good, 2007). It is important to note that the recently realized heterogeneity of this population may explain the complexity in their function.

Persistent memory responses

An important property of memory B cells is their ability to be long-lived. The ability to survive in the peripheral blood after activation allows the host to rapidly respond to a recurrent challenge in both kinetics and strength of response; as these cells have already undergone affinity maturation and are primed for quick response (Engels et al., 2009). Remarkably, these long-lived memory B cells have the potential to be sustained in the peripheral blood for decades following initial exposure to an immunological insult. The generation of long-lived memory is the cornerstone of modern vaccination. Arguably, the one of the greatest victories in the field of vaccination was the eradication of the smallpox virus in 1980 (Pennington, 2003). This feat was made possible by the life-long immunity to the virus created by the vaccine. Studies analyzing the human response to the vaccine have shown that childhood vaccination to the disease allows the patient to produce smallpox specific B cells decades after the last appearance of the disease in nature (Crotty et al., 2003, 2004; Hammarlund et al., 2003). These studies demonstrate the persistence of a proper vaccine response even after the antigen has long since disappeared from the earth (Crotty et al., 2003, 2004; Hammarlund et al., 2003). Long-lived CD27⁺ memory B cell responses have also been seen for Varicella Zoster virus and measles virus decades following initial vaccination (Pinna et al., 2009).

It has been theorized that a persistent population of specific memory B cells could lead to long-lived antibody titers through the stimulation of these memory cells by polyclonal means, such as CpG DNA and T cell cytokines; suggesting that a response to a polyclonal stimulus can cause the polyclonal activation of memory cells each with their own unique specificity, replenishing the plasma cell populations sporadically over the course of a lifetime without the requirement of specific antigenic stimulation (Bernasconi et al., 2002). If this theory is true it would help to dovetail the two ideas of long-lived memory B cells that do not require antigenic stimulation, and plasma cells, which are producing protective antibody titers over the course of a lifetime of the host; pointing to the importance of driving not only plasma cell formation but long-lived memory B cells for proper vaccine efficacy.

Memories blur

Interestingly, other common vaccinations, such as the influenza vaccination, have been seen to provide mixed results in the generation of long-term B cell memory. While the efficacy of the vaccine is dependent on the correct design of the yearly cocktail and immunity to future strains is dependent on antigenic drift and antigenic shift of the virus, CD27⁺ memory B cell persistence to the virus seems to be variable depending on the vaccinating strain (Ahmed et al., 2007; Pinna et al., 2009). Nonetheless, through analysis of the variable gene repertoire we have noted that plasmablasts induced immediately after influenza vaccination (Wrammert et al., 2008a,b) or infection (Wrammert et al., 2011) appear to be predominantly activated from pre-existing memory cells. The antibody efficacy was profoundly affected by this re-induction of memory B cells in that the antigenically similar annual strains predominantly activated B cells to epitopes that were conserved but not protective because selective pressure had induced these protective epitopes to drift (Wrammert et al., 2008a,b). Conversely, infection with the highly divergent pandemic influenza strain in 2009 activated memory cells as well, but in this case they were binding to conserved epitopes that were present because they are critical for viral function or virulence, leading to a predominantly protective response (Wrammert et al., 2011). These observations demonstrate that by targeting particular memory cells for reactivation, a better influenza vaccine might be developed that could protect against most or all influenza strains.

Reports have indicated the discovery of influenza-specific memory B cells to the 1918 pandemic flu almost 100years following initial exposure to the virus (Yu et al., 2008). Conversely, work on memory responses to influenza vaccination to A/Texas/36/91 Hemagglutinin (HA) were assayed in several patients from 1993 to 2006 and showed that levels of memory B cells specific to this viral HA were ultimately lost by their final time point (Pinna et al., 2009). It is difficult to say what might be attributing to this difference, it could be the variable conditions between live infection and vaccination or it could be a condition of constant exposure to slightly different strains each year.

Currently, work has been very limited in regard to the persistence of influenza-specific memory, but there have been studies on the elderly populations’ responses to novel 2009 pandemic H1N1 (Sasaki et al., 2011). Epidemiological data collected from the 2009 pandemic that indicated that the best protected population against the pandemic influenza were born prior to 1957; being born after this time point is associated with decreasing protection as births approach present day (Fisman et al., 2009). In a recent study, elderly populations were examined for their specific responses to either their vaccinating antigen, 2009 seasonal H1N1, or to the non-vaccinating antigen, novel 2009 pandemic H1N1 (Sasaki et al., 2011). It was seen that both groups responded equally well to the vaccinating antigen, but only the elderly patients showed increased cross-reactivity to a non-vaccinating pandemic H1N1 strain (Sasaki et al., 2011). It is thought that these differences, both epidemiologically and experimentally, derive from elderly populations’ increased exposure to influenza viruses over the course of their lives, leading to more robust memory B cell response (Fisman et al., 2009; Sasaki et al., 2011).

These recent studies provide insights into how our bodies maintain memory B cell populations over the course of time in the presence of constant antigen exposure, as in the case of influenza, or when antigen is no longer present, as with the smallpox vaccine.

This work was supported by National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (NIAID) grant numbers: 2U54AI057158-06, 2U19AI057266-06, 5U19AI062629-07, 1U19AI082724-02, 1RC4AI092711-01, and U19AI09023-01. This work was also supported by the National Institutes of Health (NIH) and National Institute of Allergy and Infectious Diseases (NIAID) Immunology Training Grant number 5T32AI007090-33.