key: cord-0905893-7itfx68w
authors: Pupovac, Aleta; Good-Jacobson, Kim L
title: An antigen to remember: regulation of B cell memory in health and disease
date: 2017-03-17
journal: Curr Opin Immunol
DOI: 10.1016/j.coi.2017.03.004
sha: db6ccba64f3fd1e99b574afd7b192d607e486c59
doc_id: 905893
cord_uid: 7itfx68w

Vaccine success relies on the formation of immunity. Humoral immunity is critical and is mediated by long-lived antibody-secreting cells and memory B cells (MBCs). Chronic infectious diseases cause a significant global burden of disease; pathogens that evade the immune system can cause phenotypical and functional changes to immune memory populations. Thus, recent studies have focused on MBC subset function. IgM(+) MBCs have emerged as important early responders in malaria. Atypical MBCs have functional qualities associated with exhaustion in chronic infectious diseases, but the requirements for their formation and where they localize remains unknown. Similarly, the T-bet-driven transcriptional program drives formation of MBCs phenotypically similar to atypical MBCs. Identifying protective or detrimental roles of MBC subsets, and their regulators, will be important for clinical intervention.

Infectious diseases are responsible for a significant global burden of disease [1] [2] [3] [4] [5] . These include human immunodeficiency virus (HIV) (which causes acquired immune deficiency syndrome (AIDs)), malaria, tuberculosis [3] , neglected tropical diseases (caused by protozoan parasites, parasitic worms and animal-borne viruses) [4] and newly emerging infectious agents (including severe acute respiratory syndrome-coronavirus, Middle East respiratory syndrome-coronavirus, West Nile virus, Nipah virus, methicillin-resistant Staphylococcus aureus, novel influenza-A strains and the Ebola virus) [5] . Diseases such as HIV/AIDS, malaria and tuberculosis are particularly more pronounced in low socio-economic regions of developing countries [1, 2] and high rates of co-infection cause additional obstacles to successful clinical intervention [1] .

Protective immunity depends on the longevity and rapid responsiveness to antigen of immune memory populations [6] . In the humoral arm of the immune system, immune memory consists of long-lived antibody-secreting plasma cells and memory B cells (MBCs). These high-affinity populations are generated during the initial immune response to infection and are mainly generated within the germinal center (GC), although they can also be generated in GC-independent pathways. Vaccines replicate this process without the threat replicating pathogens pose to the body. While vaccines have been the most effective intervention for many infectious diseases, there are still pathogens that have evaded successful vaccine design [7] . This can be the result of the genetic diversity of the pathogen, immune subversion and immunosuppression, and inconsistencies between local and systemic responses, for example, where a vaccine is not targeted to generating a mucosal immune response that may be critical for successful protection. In particular, persisting or recurrent pathogens such as HIV and Plasmodium are linked to an inadequate humoral response, in which the production of high-affinity neutralizing antibodies is delayed and the immune memory repertoire appears to be ineffective.

MBC population heterogeneity provides the functional diversity needed for the immune system to fight infection and disease [6] . MBC subsets can be segregated based on B cell receptor (BCR) isotype, phenotype, and distinct functional responses in health or immune disorders. Disease can drive alterations to MBC phenotype and function, which in turn influence antibody responses to vaccination [6] . The characteristics and regulators that shape the role of MBC subsets in infection and disease remain to be elucidated, and delineating these will provide insight into whether MBCs are beneficial or detrimental in different disease states. This review will focus on dissecting the diversity and roles of MBCs currently trending in the literature, including immunoglobulin (Ig) M + , atypical and T box transcription factor (T-bet) + MBCs, and their roles during infectious disease. balance self-renewal with differentiation during a secondary response. Classically, MBCs have been distinguished by their Ig isotype: IgM + or class switched (IgG + IgA + and IgE + ) [6] , but they can also be distinguished into subsets by surface markers such as CD73, CD80 and PD-L2 [9 ] . MBC function had been defined by these subdivisions, for example, IgM + MBCs proliferate and form secondary GCs for self-renewal and longevity of the MBC population, whilst IgG + MBCs differentiate to become antibody-secreting cells [6] ( Figure 1, top panel) . This distinction, however, is not absolute. It appears that the maturity of an MBC is the main predictor of whether it will reenter GCs or differentiate into an antibody-secreting cell during a secondary response, which correlates but is not absolutely dependent on Ig isotype. It is now evident that IgM + MBCs can be both GC-derived and GC-independent, have unique phenotypes and functional properties compared to switched MBC, and persist to provide long-term memory [9 ,10,11] .

While recent research has focused on defining subsets in vivo using immunization models, the role of IgM + MBCs in chronic and recurrent disease remains ill defined. To this end, Pepper and colleagues produced B cell tetramers specific for blood stage Plasmodium chabaudi antigen, merozoite surface protein 1 (MSP1), to study antigenspecific MBCs in a model of malaria. MSP1-specific IgM + MBCs were comparable to switched MBCs with respect to phenotypic expression of CD80 and CD73, their somatic hypermutation rates, affinity and function [12 ] . Interestingly, MSP1-specific MBCs were early responders, persisted and were dominant producers of antibody-secreting cells during secondary responses [12 ] , suggesting that these cells play a protective role during reinfection ( Figure 1 , middle panel). Further, they 90 Lymphocyte development and activation were able to form antibody-secreting cells in the absence of T cell help. Genetic resistance to malaria has been correlated with enhanced IgM responses compared to IgG responses [8] , also suggesting a possible role for IgM + MBCs in long term protection from malaria. Given the propensity of IgM + MBCs to generate new GCs [10, 11] , IgM + MBCs may also be important for expanding the MBC repertoire to target diversifying antigens.

By providing the ability of GC re-entry, the option to renew the repertoire allows for flexibility to contain fast evolving antigens. However, if a mutation disrupts this process it may lead to development of disease. In this regard, in a chronic infection model where MBCs constitutively express B cell lymphoma 2 (BCL2) and contain the follicular lymphoma hallmark, IgH/BCL2 (t(14;18) (q32;q21)) translocation, IgM + MBCs are able to re-enter GCs repeatedly upon chronic immunization, leading to and driving follicular lymphomagenesis [13 ] (Figure 1 , lower panel). This finding reveals a novel role for IgM MBCs in cancer development and progression. A number of B cell leukemias and lymphomas express IgM and resemble MBCs in terms of phenotype and genetics [14] . It is unknown why IgM is commonly expressed, but considering IgM + MBCs are poised to proliferate upon activation in comparison to switched MBCs, IgM + MBCs may be at an increased risk to transform into constantly proliferating lymphomas [14] .

Atypical MBCs are associated with exhaustion in chronic or recurrent infectious disease MBC phenotype and function appears to be correlated with the nature of the infectious agent. In particular, the emergence of atypical or unresponsive MBCs has been associated with the presence of persistent antigen [15, 16] . Several studies have described the presence of an atypical MBC subset that is prominent in chronic infections including human immunodeficiency virus (HIV) [16, 17] , malaria [15,18 ,19,20] , HIV/malaria co-infected patients [21] , hepatitis C [22] and cytomegalovirus infection [23] . These atypical MBCs are reportedly functionally impaired, often referred to as 'exhausted', due to the upregulation of inhibitory receptors, modulation of tissue trafficking receptors and poor proliferative abilities and antibody responses [15,16,18 ,19] . HIV-specific responses are enriched in functionally impaired atypical MBCs, suggesting that these cells are unable to produce antibody to target HIV antigens, thus explaining the poor HIV-specific antibody response in individuals [16] . This study was confirmed at a single cell level, using a trimeric HIV envelope probe, which directly showed that HIVspecific responses were enriched in an activated MBC subset prone to apoptosis [17] . Although a similar subset has been characterized in malaria patients, it is unclear whether atypical MBC are dysfunctional in malaria patients and thus whether they can be defined as the same subset as that found in HIV patients. A number of studies provide evidence of atypical dysfunctional MBCs in malaria [18 ,19,20] , although a conflicting study showed that atypical memory subsets could proliferate and secrete antibody [24 ] . While the latter study suggested atypical MBCs might originate from different precursors due to differences in their somatic hypermutation loads and clonal relationship, others have shown a common developmental history [19, 25] . This discrepancy may be related to the large diversity of antigens in this disease. The majority of studies to date did not profile antigen specificity of atypical MBCs, making it difficult to fully elucidate their biological roles in disease. B cell tetramers targeted for different antigens will be useful in future studies of phenotypes and functions of antigenspecific MBCs in responses to persisting or recurrent pathogens that can lead to chronic disease.

The markers that are used to distinguish atypical MBCs from other MBC subsets include downregulation of CD27 and CD21 [15-17,18 ,19-23,24 ,25,26] . Atypical MBCs and other CD21 neg B cell subsets [27] [28] [29] [30] [31] [32] are enriched in the peripheral blood of patients with diseases involving chronic immune stimulation [33] . While CD21 neg subsets tend to differ in the expression of CD27 and BCRs in different diseases, atypical and CD21 neg subsets exhibit common expression of many inhibitory and cell activation receptors that are associated with exhaustion and anergy (Table 1 ) [33] . Commonalities across subsets includes the increased expression of CD11c and CXCR3, which are molecules involved in homing to sites of inflammation and reduced expression of CCR7, CD62L, CXCR4 and CXCR5 which are required for migration to spleen or lymph nodes (Table 1) . This suggests that atypical MBCs and CD21 neg B cells migrate to sites of inflammation. Fc-receptor like (FCRL) receptors, in particular FCRL4, was also commonly expressed on these subsets (Table 1) . FCRL4 MBCs were first described as tissue-localized memory cells in healthy individuals [34] , and are speculated to define atypical MBCs in malaria [15, 24 ] and HIV [16] ( Figure 2) . Other more recent studies suggest that functionally impaired atypical memory in malaria is delineated by the expression of FCRL5 rather than FCRL4 [18 ,19,35] , which may be due to cross reactivity of FCRL4 antibodies used in previous studies [18 ] , a factor that needs to be addressed in future studies.

The role of atypical MBCs in tissue localization is undefined in disease and warrants investigation, given that a recent study suggested tissue localization could provide broader protection from highly mutating viruses [36 ] . MBCs residing at the site of influenza infection develop from persistent GCs in the lung, are highly mutated, and have cross-reactive antibody responses that neutralize flu escape variants [36 ] . Defining the pathways that result in cross-reactivity may have significant implications for the creation of broadly protective vaccines against highly mutating pathogens and may help to shed light on discrepancies between systemic versus localized responses to vaccines. Furthermore, characterizing migration to and out of tissues will shed light on intrinsic and extrinsic regulators of MBCs during chronic infection. For example, IFN-I production in chronic lymphocytic choriomeningitis virus (LCMV) infection recruits T cells, monocytes and dendritic cells to directly act on antigenspecific B cells and thus inhibit LCMV-neutralizing antibody responses [37] [38] [39] . Currently there are no in vivo models of atypical memory, yet understanding migration and localization of both classical and atypical memory will be important in determining targets for clinical intervention.

Transcription factors are essential in regulating B cell differentiation [40] . B cell-specific T-bet expression is essential for IgG2a/c MBC survival [41] , drives interferon g (IFNg)-mediated IgG2a/c class switching and upregulates CXCR3 expression on MBCs to allow migration to sites of inflammation [42] . In chronic LCMV infection, B cell-specific T-bet controls IgG2a/c production and is required to contain persistent infection [43 ] (Figure 3) , as well as a transcriptional program that affects cell migration, localization, differentiation, proliferation and antibody development compared to T-bet À cells [43 ] . In particular, T-bet + MBCs express high levels of CXCR3 (Figure 3) , which is also expressed on atypical MBCs [15, 16, 23] and other CD21 neg B cell subsets [33] (Table 1) . Consistent with CXCR3 expression, T-bet MBCs have been found in small intestinal mucosa [43 ] , which is a site of inflammation induced by chronic LCMV infection [44] . This suggests that T-bet is involved in the control of multiple immune functions including tissue localization during chronic infection [43 ] . In this regard, T-bet may be involved in the formation and function of MBCs that reside at the site of influenza infection and have cross-reactive antibody responses that neutralize flu escape variants [36 ] . While there is a lack of studies examining the role of T-bet on atypical MBCs; one study has shown high expression of the T-bet gene, Tbx21 in FCRL5 + cells in healthy individuals [35] . Furthermore, T-bet is reported to regulate CD8 + T cell exhaustion in HIV infection [45] . Revealing a role for T-bet in formation and/or function of atypical MBCs may prove additional insight into dysfunctional humoral responses.

helper cell-associated cytokines, IL-21, IFN-g and IL-4 [46 ] . In particular, T-bet + CD11c + MBCs are promoted in IL-4-low environments. For example, after infection with influenza (which induces IL-21, IFN-g and IL-4 microenvironments), T-bet + CD11c + MBCs are present in large quantities in IFN-g and IL-4 double knockout mice, but not in IFN-g knockout mice (Figure 3 ). In helminth infection (IL-21 and IL-4 present), T-bet + CD11c + MBCs are present in IL-4 knockout mice but not in wild-type mice (Figure 3 ). TLR7 activation also induces a CD11c + subset in age-associated or autoimmune-associated B cells [28] . Interestingly, transcript profiling of this subset revealed high expression of T-bet [28] (Figure 3 ). This subset is CD21 neg , has blunted BCR responses (similar to atypical MBCs), Table 1 Inhibitory and activation receptor/gene expression on atypical MBCs and CD21 neg B cells. Inhibitory and activation receptor/gene expression ("-upregulation and #-downregulation) is shown compared to classical MBCs, naive or CD21 + B cells

Receptor/gene expression Reference Malaria CD11c ", CD22 ", CD72 ", CD85j ", CD200R1 ", CXCR3 ", FCGR2B ", FCRL3 ", FCRL4 ", FCRL5 ", LILRB1 ", LILRB2 ", SIGLEC-6 ". [20,23,24 ,29] CCR7 #,CD62L #, CXCR5 #.

Human immunodeficiency virus CD11c ",CD22 ", CD72 ", CD85j ", CXCR3 ", CCR6 ", FCGR2B ", FCRL4 ", LILRB1 ", LILRB2 ", SIGLEC-6 ". [20, 22] CCR7 #, CD62L #, CXCR5 #.

CD11c ", CD22 ", CD72 ", CD85j " CXCR3 ".

[23] CCR7 #, CD62L #, CXCR4 #, CXCR5 #.

CD11c ", CD22 ", CD72 ", FCRL2 ", FCRL3 ", SIGLEC ".

[31] CD1c #.

CD11c ", CD72 ", CRL2 ", CXCR3 ", CXCR6 ", FCGR2B ", FCRL3 ", FCRL5 ", FCRLM1 ", FCRLM2 " LILRB ", SIGLEC " SOX5 ".

[32-34]

Hepatitis C associated-mixed cryoglobulinemia CBLB ", CD22 ", CD72 ", CD200R1 ", EGR2 ", FCRL4 ", LAX1 ", LGALS1 ", Stra13 ", ZEB2 ". accumulates in aged female mice, forms early in female autoimmune prone mice and is present in patients with rheumatoid arthritis [28, 47] . Furthermore, this subset directly contributes to the production of autoantibodies and therefore may contribute to autoimmunity [28] ( Figure 3 ). CD11c is also highly expressed on atypical MBCs, other CD21 neg subsets (Table 1 ) [15,16,19,20,24 ,26,28,29] , and in FCRL4 + MBCs that may play a role in mucosal defense [48] . Interestingly, many CD21 neg subsets in disease also express IgM [33] . Determining the relationship between TLR engagement, T-bet activation, CD21, CXCR3, CD11c and IgM in MBCs may reveal mechanistic commonalities in dysfunctional humoral disorders that are characterized by aberrant immune stimulation.

The common expression of inhibitory, migration and activation markers on atypical MBCs (Table 1 ) and in T-bet + MBCs present in chronic infection, aging and autoimmunity suggests that a T-bet-driven transcriptional program may regulate the formation and/or function of atypical memory, in addition to age/autoimmune-associated subsets. Several questions arise: Is T-bet involved in tissue B cell memory subsets in health and disease Pupovac and Good-Jacobson 93 

In diseases involving highly diversifying antigen, MBC heterogeneity may be central in containing chronic infection. IgM + MBCs have emerged as important early responders in malaria re-infection and therefore may be vital in early parasite clearance in humans. Whether IgM + MBCs function as early responders in other chronic infections remains to be determined. Atypical MBCs and other CD21 neg subsets are exhausted in several different chronic infectious diseases, but how these different diseases affect this subset and how they are formed and function within these diseases remains unknown. Targeting commonly expressed inhibitory and activation markers may provide a therapeutic avenue to ameliorate putative detrimental effects of atypical MBCs. Short inhibitory RNA knockdown of inhibitory receptors on atypical MBCs in HIV enhanced BCRmediated proliferation, in particular the largest effect was observed when targeting FCRL4 and sialic acidbinding immunoglobulin-type lectin 6 (Siglec-6) [49] . These and other commonly expressed receptors may be potential targets for receptor antagonists to reactivate MBCs and induce protective antibody responses. Lastly, T-bet-driven transcriptional regulation may be involved in the formation and function of atypical MBCs and age/autoimmune-associated subsets. Identifying the characteristics and regulators of MBC heterogeneity in the context of chronic infection, and the impact on the generation of classical memory, will be important for innovative solutions to eradicate these diseases.

The authors declare no conflict of interest. 

Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest

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Modeling infectious disease dynamics in the complex landscape of global health

Global, regional, and national incidence and mortality for HIV, tuberculosis, and malaria during 1990-2013: a systematic analysis for the Global Burden of Disease Study

Neglected tropical diseases: diagnosis, clinical management, treatment and control

Emerging infectious diseases: threats to human health and global stability

Memory B cells

Editorial: why vaccines to HIV HCV, and malaria have so far failed-challenges to developing vaccines against immunoregulating pathogens

Multiple routes to B-cell memory

CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype

Using a B cell transfer system, these authors showed that cell surface markers other than IgM and Ig class-switch could segregate memory B cell subsets and their function

Different B cell populations mediate early and late memory during an endogenous immune response

Multiple layers of B cell memory with different effector functions

Somatically hypermutated plasmodium-specific IgM + memory B cells are rapid, plastic, early responders upon malaria rechallenge

Using a tetramer, these authors demonstrated the functional role of IgM memory B cells in immune responses to malaria

Germinal center reentries of BCL2-overexpressing B cells drive follicular lymphoma progression

Human memory B cells

Atypical memory B cells are greatly expanded in individuals living in a malaria-endemic area

Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals

Abnormal B cell memory subsets dominate HIV-specific responses in infected individuals

FCRL5 delineates functionally impaired memory B cells associated with Plasmodium falciparum exposure

This authors demonstrate that FCRL5 was associated with dysregulated memory B cell s in malaria

Malariaassociated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function

Chronic exposure to Plasmodium falciparum is associated with phenotypic evidence of B and T cell exhaustion

HIV malaria co-infection is associated with atypical memory B cell expansion and a reduced antibody response to a broad array of Plasmodium falciparum antigens in Rwandan adults

Peripheral CD27 À CD21 À B-cells represent an exhausted lymphocyte population in hepatitis C cirrhosis

Primary human cytomegalovirus infection induces the expansion of virus-specific activated and atypical memory B cells

Atypical and classical memory B cells produce Plasmodium falciparum neutralizing antibodies

This study concluded that atypical memory B cells were functionally capable to produce neutralizing antibodies, in contrast to other studies of malaria

The V gene repertoires of classical and atypical memory B cells in malaria-susceptible West African children

Expansion of autoreactive unresponsive CD21 À/low B cells in Sjogren's syndrome-associated lymphoproliferation

Complement receptor 2/CD21-human naive B cells contain mostly autoreactive unresponsive clones

Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c + B-cell population is important for the development of autoimmunity

Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells

Clonal B cells in patients with hepatitis C virus-associated mixed cryoglobulinemia contain an expanded anergic CD21 low B-cell subset

Expansion of functionally anergic CD21-/low marginal zone-like B cell clones in hepatitis C virus infectionrelated autoimmunity

Clonal B cells of HCV-associated mixed cryoglobulinemia patients contain exhausted marginal zone-like and CD21 low cells overexpressing Stra13

Martensson IL: CD21 À/low B cells: a snapshot of a unique B cell subset in health and disease

Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells

Fc receptor-like 5 expression distinguishes two distinct subsets of human circulating tissue-like memory B cells

Distinct germinal center selection at local sites shapes memory B cell response to viral escape

This study demonstrated the importance of tissue localisation for memory B cells in responding effectively to influenza

Type I interferon suppresses virus-specific B cell responses by modulating CD8+ T cell differentiation

Inflammatory monocytes hinder antiviral B cell responses

Interferon-driven deletion of antiviral B cells at the onset of chronic infection

Regulation of germinal center B-cell memory, and plasma cell formation by histone modifiers

Divergent transcriptional programming of class-specific B cell memory by T-bet and RORa

CD8 T cells induce T-bet-dependent migration toward CXCR3 ligands by differentiated B cells produced during responses to alum-protein vaccines

Cutting edge: B cell-intrinsic T-bet expression is required to control chronic viral infection

Intestinal intraepithelial lymphocytes respond to systemic lymphocytic choriomeningitis virus infection

T-bet and Eomes are differentially linked to the exhausted phenotype of CD8 + T cells in HIV infection

Cutting edge: IL-4, IL-21, and IFN-g interact to govern T-bet and CD11c expression in TLR-activated B cells

This study demonstrated the importance of balancing different cytokines in the microenvironment to drive appropriate B cell transcriptional programs

A B-cell subset uniquely responsive to innate stimuli accumulates in aged mice

Discriminating gene expression profiles of memory B cell subpopulations

Attenuation of HIV-associated human B cell exhaustion by siRNA downregulation of inhibitory receptors

This work was supported by a National Health and Medical Research Council (NHMRC) project grant (1057707) and an NHMRC Career Development Fellowship to KLG-J.