*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Immunological Memory, a section of the journal Frontiers in Immunology*

Received: *14 February 2019* Accepted: *03 June 2019* Published: *02 July 2019*

#### Citation:

*Auladell M, Jia X, Hensen L, Chua B, Fox A, Nguyen THO, Doherty PC and Kedzierska K (2019) Recalling the Future: Immunological Memory Toward Unpredictable Influenza Viruses. Front. Immunol. 10:1400. doi: 10.3389/fimmu.2019.01400* Persistent and durable immunological memory forms the basis of any successful vaccination protocol. Generation of pre-existing memory B cell and T cell pools is thus the key for maintaining protective immunity to seasonal, pandemic and avian influenza viruses. Long-lived antibody secreting cells (ASCs) are responsible for maintaining antibody levels in peripheral blood. Generated with CD4+ T help after naïve B cell precursors encounter their cognate antigen, the linked processes of differentiation (including Ig class switching) and proliferation also give rise to memory B cells, which then can change rapidly to ASC status after subsequent influenza encounters. Given that influenza viruses evolve rapidly as a consequence of antibody-driven mutational change (antigenic drift), the current influenza vaccines need to be reformulated frequently and annual vaccination is recommended. Without that process of regular renewal, they provide little protection against "drifted" (particularly H3N2) variants and are mainly ineffective when a novel pandemic (2009 A/H1N1 "swine" flu) strain suddenly emerges. Such limitation of antibody-mediated protection might be circumvented, at least in part, by adding a novel vaccine component that promotes cross-reactive CD8+ T cells specific for conserved viral peptides, presented by widely distributed HLA types. Such "memory" cytotoxic T lymphocytes (CTLs) can rapidly be recalled to CTL effector status. Here, we review how B cells and follicular T cells are elicited following influenza vaccination and how they survive into a long-term memory. We describe how CD8+ CTL memory is established following influenza virus infection, and how a robust CTL recall response can lead to more rapid virus elimination by destroying virus-infected cells, and recovery. Exploiting long-term, cross-reactive CTL against the continuously evolving and unpredictable influenza viruses provides a possible mechanism for preventing a disastrous pandemic comparable to the 1918-1919 H1N1 "Spanish flu," which killed more than 50 million people worldwide.

Keywords: T cells, B cells, influenza, immunological memory, vaccine

# INTRODUCTION

Successful vaccination relies on the induction of long-term immunological memory. Exposure to an infectious virus elicits acute effector responses that mediate acute pathogen control, along with the generation and maintenance of T cell and B cell memory capable of protecting against re-exposure. At sufficient levels, neutralizing antibody (Ab) can prevent reinfection while, especially if such protection is partial, the rapid recall of memory CD8+ cytotoxic T lymphocytes (CTLs) facilitates enhanced pathogen control. Seasonal influenza results from the emergence of an occasional, highly infectious variant selected as a consequence of Ab-driven mutational change in the viral envelope hemagglutinin (HA) and/or neuraminidase (NA) proteins. Pandemic influenza A viruses, on the other hand, arise from gene reassortment of two different influenza A virus (IAV) subtypes infecting the same cells. As a consequence, the influenza research and control community face the continuing challenge of producing new vaccines to control emerging threats.

Most of the existing products utilize inactivated virus, or isolated viral HA and NA proteins, that stimulate influenza strain-specific antibody immunity and B cell memory, but do not prime the much more cross-reactive CD8+ CTL compartment. The challenge is thus to add a T cell-targeted vaccine component that promotes CTL memory for the rapid recall of anti-viral CTL effectors to the respiratory tract for early virus control and/or induce cross-protective B cells. In this review, we focus on the nature of optimal memory B cell and T cell generation and ask how we might use this knowledge to overcome the limitations of seasonal influenza vaccines by developing feasible strategies for both inducing and maintaining long-term, cross-reactive immunological memory.

#### The Burden of Seasonal Influenza

Seasonal influenza virus is a global health problem. In the United States, influenza virus infections causes 9.2–35.6 million cases of illness, 140,000–710,000 hospitalizations and 12,000– 56,000 deaths per year (1). Globally, it is estimated that every year 290,000–650,000 respiratory deaths are due to seasonal influenza (2). The World Health Organisation (WHO) recommends annual influenza vaccination for people at high risk of developing severe disease, and for those in contact with high-risk individuals. Vulnerable groups include the elderly (>65 years), young children (6–59 months), Indigenous populations, patients with chronic medical conditions, pregnant women, and health-care workers (3). National health authorities in the countries with an advanced public health system recommend annual vaccination for everyone 6 months of age and above, both to protect individuals and to limit the spread of the virus through the community (4, 5).

### Influenza Virus Evolution Poses a Challenge for Long-Term Humoral Immunity and Vaccine Effectiveness

Influenza viruses attach to host cells via HA binding to cell surface sialic acids (6, 7). Protective antibodies (Abs) block virus attachment by binding to the antigenic sites (8–11) proximate to the sialic acid receptor binding pocket on the HA head. Such Abs are the best correlate for influenza immunization and are measured using the hemagglutination inhibition (HAI) assay, which detects Abs blocking the capacity of the virus to agglutinate red blood cells by binding to sialic acids on their surface (12). The influenza virus RNA polymerase lacks proof-reading function, with the consequence that there is a constant emergence of mutants (affecting viral fitness and/or immune recognition) carrying substitutions that arise randomly across the genome. Antibody-mediated immune pressure drives the selection of viruses expressing variant HAs and NAs (13, 14) that, if their "fitness" is not unduly compromised, have the potential in nature to cause the process that has long been called antigenic drift (15, 16). Clearly, for a drifted strain to emerge as a clinical problem, its HA must be sufficiently changed to escape neutralization by pre-existing antibodies induced broadly in human populations by past infections and/or vaccinations. The reality that individuals who were once protected are now at risk from the new variant strain is the basis for frequently reformulating seasonal influenza vaccines (17). In contrast, through the process of antigenic shift, influenza viruses incorporate a completely new HA or NA (18), which adds a new virus into the epidemiological mix. When it comes to antibody-mediated selection, the A/H3N2 strains have consistently shown the greatest antigenic drift for the three types of influenza viruses that co-circulate globally and cause seasonal epidemics (A/H1N1, A/H3N2, and influenza B viruses) (16, 19). In general, more extensive epidemics (with increased morbidity and mortality) occur when a novel, seasonal A/H3N2 drifted strain emerges (16, 20, 21).

Multi-component influenza vaccines are designed to elicit serum antibodies against the HAs of one A/H1N1 strain, one A/H3N2 strain and one (or two) influenza B viruses (Yamagata or Victoria) (22). Increased antibody titres induced by vaccination decrease the risk for infection caused by any strains antigenically similar to those included in the vaccine (23, 24), although they confer limited or no protection against other types or subtypes (including drifted variants) of influenza (25). The global WHO network closely monitors the circulation of influenza viruses in humans and other species, including birds, across the northern and southern hemispheres, whereby information derived from the antigenic and genetic characterization of these strains, along with epidemiological data, is used to select the strains to be incorporated into an upcoming seasonal vaccine (26). This strategy can fail, at least in part, as vaccine preparation takes at least 6 months and the product may no longer match all 3 (or 4) circulating viruses by the time it is released (27). Moreover, pre-existing immunity in humans can be highly variable due to age and prior exposures via infection and/or vaccination (28– 34). The level of pre-existing human immunity is considered but often difficult to interpret due to high heterogeneity. Firstinfection ferret antisera is used to identify and characterize new influenza strains, yet repeated exposures to A/H3N2 variants affect Ab quantity and quality, which makes vaccine-strain selection even more challenging (35). Both immunological responses to influenza viruses and influenza vaccine effectiveness are undoubtedly affected by the combination of antigenic drift and prior immunity. Influenza virus evolution has been widely studied, yet it is still largely unknown how cross-reactive B cell memory impacts on Ab responses to new strains.

# B Cell Memory and Imprinting Against Prior Strains

The idea that immunological memory could impact negatively on Ab responses to novel influenza strains first emerged in the early 1950s, when Francis and Davenport observed that the exposure to a new influenza strain induced higher titres of Abs against variants encountered in childhood than against the prevailing strain (36–39). They proposed the colorfully named concept of "original antigenic sin" (OAS), which states that Abs generated against the first antigen (Ag) encountered in childhood would be repeatedly and preferentially induced at every exposure, even if the epitope remained as a minor secondary antigen. This was considered to be sinful, i.e., detrimental for protection against following influenza infections, since the Abs induced poorly neutralized the most recent strain that had actually triggered them (40, 41). Molecular level analyses of B cell receptor usage have since confirmed that memory B cells elicited by a priming Ag can participate in the immune response toward a structurally related, boosting Ag (42, 43). While it is clear that somatic mutation of the immunoglobulin (Ig) variable (V) region takes place, the extent to which this leads to increased affinity for the priming vs. boosting variant remains controversial (42). These molecular analyses are consistent with more recent observations that Ab boosting is broad, and greatest against more similar viruses, differing somewhat from the OAS concept that centers on the initial antigen encountered (44, 45). Efforts to understand why prior vaccination enhances vaccine effectiveness in some influenza seasons, yet attenuates it in others, has led to further refinements to the OAS hypothesis, namely that imprinted B cell memory responses are not inevitably "sinful" i.e., ineffectual (31). Hensley et al. propose that Ab become focused on selected epitopes which are relatively conserved between successive strains due to a form of competitive dominance by memory B cells and that while this may result in high Ab titres and clinical benefit it may, alternatively, compromise protection if the epitope is altered in future strains. This hypothesis is based on molecular and serological analyses that document focused HI Ab responses in selected individuals (29, 30, 46–49).

At the cellular level, it is clear that memory B cells respond more rapidly than their naïve precursors. Hence, antibody responses may become focused on epitopes that were present in earlier strains because memory B cells specific for those epitopes become rapidly activated at the expense of naïve B cells, which need a higher threshold to respond (50, 51). Memory B cells that bear affinity matured antigen receptors may also be better able to compete with existing Abs for inducing antigen than naïve B cells (52). Several strategies have the potential to promote naïve B cell activation and broaden the Ig response. These include giving repeated vaccine doses (39), increasing the amount and concentration of antigen (53), and adding adjuvants (54). Another suggested mechanism that may promote the enhanced engagement of memory (vs. naïve) B cells is that T regulatory cells (Tregs) induced by the initial encounter reduce the amount of antigen presented on dendritic cells, thus diminishing the antigen availability for naïve B cells, promoting a memory B cell boost at the expense of naïve B precursors (55).

# Current Strategies to Improve Seasonal Influenza Vaccine Effectiveness

Strategies to increase seasonal influenza vaccine effectiveness (VE), like high-dose or adjuvanted vaccines, are still under evaluation. Pooled analysis of multiple studies showed that high-dose vaccines significantly reduce the risk of laboratoryconfirmed influenza cases in the elderly when the vaccine and the circulating strains are well-matched, but not when they are mismatched. The HAI geometric mean titres after vaccinating with the high-dose vaccine were significantly higher compared to the standard-dose vaccine for the H3 component. However, the proportion of participants with seroprotective HAI Ab levels (HAI titer ≧ 1:40 or 1:32) was the same using both vaccines (56). Similarly, high-dose vaccines showed significant increases in VE with a reduction in mortality among the elderly by 36.4% in the 2012–2013 season, when H3N2 viruses were predominantly circulating (57). Nonetheless, seasonal VE on that season was only of 11% for that particular age group (58), indicating that a high-dose vaccine, despite increasing VE, did not induce an epidemiologically significant improve in overall H3N2 VE. Alternatively, the use of a standard-dose influenza vaccine with the MF59 adjuvant (Novartis) can reduce laboratory-confirmed influenza cases as well as hospitalizations due to influenza in the elderly (59) and seasonal trivalent vaccines formulated with this adjuvant are now available for those >65 years old (FluAd, Sequiris).

In addition to MF59, other adjuvants licensed for use with inactivated or sub-unit-based influenza vaccines include Alumcontaining formulations (AlPO4 or Al[OH]3) and oil-in-water emulsions, AS03 (GSK) and AF03 (Sanofi Pasteur). The benefits of using these adjuvants to increase seroprotective antibody titres are widely reported in a number of clinical studies, including in individuals who are most susceptible to influenzarelated illness. Compared to non-adjuvanted vaccine responses, formulation of mono- and multi-valent influenza vaccines with MF59 induces substantially higher HAI titres and seroconversion rates in children (60–63) with similar improvements observed in the young and elderly using AS03 (64). These formulations are generally well-tolerated and safe, however, incidences of narcolepsy associated with the use of an AS03-adjuvanted A/H1N1pdm2009 vaccine (Pandemrix) limits the use of this adjuvant in the young. Nevertheless, both MF59 and AS03 have been shown to accelerate the induction of vaccine-mediated responses as demonstrated by the use of adjuvanted vaccines in healthy adults (65, 66), children (67) and in the elderly (68), wherein a single vaccination dose is sufficient to induce seroprotective levels of antibody within as little as 3 weeks. In this regard, these adjuvants, along with AF03 or Alum, provide dosesparing capabilities for mass vaccination of the wider population; similar levels of protection attained with unadjuvanted vaccines can be achieved with using substantially smaller amounts of HA antigen or less vaccination doses when formulated with adjuvant (69–72). Several studies have also demonstrated the ability of MF59 to induce cross-reactive antibodies against non-vaccine matched strains in prime-boost regimens. Priming of subjects with a clade 0 H5N3 vaccine formulated with MF59 followed by a boost with a clade 1 H5N1 vaccine containing the same adjuvant results in high titres of cross-neutralizing antibody against H5N1 clade 0, 1 and 2 viruses (73–75). These results thus highlight the role that adjuvants can play in generating and broadening the cross-specificity of naïve and pre-existing B cell memory, the possible underlying mechanisms of which are discussed further in subsequent sections below.

Influenza vaccines designed to target Abs toward the conserved epitopes in the HA stem are also under intense study. While heterosubtypic protection with group 1 HA stem vaccines (i.e., H1 and H5 viruses) lacking the highly variable HA head has been demonstrated in animal models (76), studies on group 2 HA stem vaccines (i.e., H3 and H7 viruses) are more limited. Although promising results are observed when immunizing mice with conserved HA stem epitopes from the H3 subtype, by way of cross-clade neutralizing activity (77, 78), immunogenicity and protection are not maintained when using larger animal models like ferrets (78). Therefore, further studies are needed to develop a human B cell-based universal influenza vaccine, with consideration into the potential for influenza viruses to escape from HA-stem targeted Abs (79).

#### Dissecting the B Cell Response

Activation of naïve B cells can elicit short-lived ASCs (also called plasmablasts), long-lived antibody-secreting plasma cells (LLPCs), and memory B cells. The fate of B cells is considered to be highly orchestrated, depending on the mode of stimulation, the affinity of their B cell receptors (BCR, or surface Ig) for antigen and their location (80–82). In the periphery, within secondary lymphoid organs (SLO), naïve B cells are activated by BCR/Ag binding and, depending on whether T cell help is provided, they will continue the response in a T celldependent (TD) or T cell-independent (TI) manner. B cell memory resulting from a TI response expresses and produces IgM capable of engaging at broadly low affinity with antigens via multivalent BCR engagement, plus toll-like receptor (TLR), and/or complement engagement (83). In TD responses, B and T cell interaction occurs when antigen is captured through the BCR of specific naïve B cells and presented via cell-surface MHC-II glycoproteins to CD4+ helper T cells specific for peptides from the same antigen (84, 85). All B cells activated in this manner either move into lymph node follicles and generate germinal centers (GCs) or differentiate into extrafollicular plasmablasts (86, 87). Through this array of processes, different classes of memory B cells are generated, which can be distinguished by their passage through the GC, location and Ig isotype (81).

In the GC, B cells undergo intense proliferation and broaden their BCR diversity through somatic hypermutation, a process whereby point mutations, insertions, and deletions are introduced within Ig V gene hotspots to generate a broad array of B cell clones with a broad spectrum of affinities for the immunizing Ag (88). This process results in the generation of memory B cells with high-affinity surface Igs and surface Ig+/− plasma cells that maintain serum immunoglobulin levels against the foreign invader. The GC is also the site where a large proportion of BCR-defined clones undergo class switch recombination (CSR), exchanging the Ig isotypes originally expressed (IgM and IgD) for IgG, IgA, or IgE (88–90). The sequential generation of long-lived memory B cells in the GC starts from unswitched memory B cells, followed by classswitched memory B cells and, finally, by LLPC that travel to the bone marrow and other sites (91). The later each B cell population appears, the higher its affinity for Ag (92). Hence, B cells with lower affinity BCRs have a greater propensity to enter, and persist in, the memory pool. Intriguingly, such memory-directed B cells show enhanced Bach2 transcription factor expression when compared to their counterparts with higher BCR affinity, and Bach2 expression inversely correlates with the strength of the B-T follicular helper (Tfh) cell interaction. This suggests that B cells with lower affinity receive weaker T cell help and express higher levels of Bach2, which is clearly a key factor in memory B cell fate determination (92). In addition, expression levels of Blimp-1, the key regulator of plasma cell differentiation and CSR, are regulated by Bach2. Higher Bach2 levels decrease Blimp-1, promoting B cell differentiation toward an unswitched memory fate. The aryl hydrocarbon receptor (AhR), a ligand-induced nuclear transcription factor, is highly induced in B cells upon BCR engagement. AhR promotes Bach2 expression, which in turn suppresses Blimp-1 and therefore the B-Tfh cell interaction becomes weaker and B cell CSR and differentiation into plasma cells are suppressed (93), indicating that it may be a potential target in promoting the generation of low-affinity IgM+ B cell memory upon vaccination. This is of particular relevance for the design of the next generation influenza vaccines since, as discussed below, as there is an increasing body of evidence suggesting that low-affinity IgM+ memory B cells capable of identifying a broad range of epitopes should be targeted by influenza vaccination.

#### Heterogenous Memory B Cell Phenotypes Have Different Roles in Secondary Responses

The various modes of TD and TI B cell activation generate memory B cells with varying isotypes and affinities (summarized in **Figure 1**), some bearing highly mutated Igs generated via the GC reaction and others maintaining germline, less specific and more cross-reactive Abs (52, 81, 94). While it is generally accepted that memory B cells show an enhanced capacity for terminal differentiation into ASC, regardless of phenotype and affinity, there is less consensus regarding their propensity to (re) enter GC reactions. Contrary to early thinking, it is now generally accepted that both IgG+ and IgM+ memory B cells can re-enter GC reactions, albeit they are more predisposed to differentiate into ASC during recall responses (52, 95–97). Similarly, whether or not GCs form during recall responses together with the character of the memory B cell subsets that participate may depend on the type and amount of antigen, inflammatory signals and the availability and quality of cognate Tfh cells (98). There is evidence that unswitched memory B cells bearing germline BCRs have a greater propensity to enter the GC reaction (99). In particular, IgM+ cells with the least mutated V genes were

FIGURE 1 | Pathways to B cell memory. Naïve B cells become activated by direct recognition of antigens expressed on the surface of the pathogen. Top panel: Follicular (FO) naïve B cells become activated within the lymph node through a T cell-dependent pathway. CD4+ T cells become activated by recognizing viral peptides processed by FO dendritic cells and presented on their surface by MHC-II molecules. After becoming activated, both CD4+ T cells and B cells, travel to the T-B border in the lymph node, where they interact. Three outcomes can follow this interaction. (i) A germinal center (GC) is formed, CD4+ T cells polarize into T follicular helper (Tfh) cells and FO B cells differentiate into GC B cells. In the GC, B cells undergo rapid proliferation and somatic hypermutation of the Ig V regions in their B cell receptors (BCR), due to their interaction with Tfh cells through CD40-CD40L, PD1-PD-L1/L2, ICOS-ICOSL among others and the secretion of cytokines such as IL-4 and IL-21, affinity maturation takes place and those B cells that increase affinity toward their Ag are selected. Some of these B cells will also class-switch. These interactions result in the generation of IgM+ memory B cells (BMEM), IgG+/A+/E+ BMEM or IgG/A/E secreting long-lived plasma cells (LLPC) in this order in time. The later these cells are generated, the higher affinity and lesser cross-reactivity they have toward the antigen or antigen variants, respectively. (ii) Not all B cells enter the GC after interacting with their cognate activated CD4+ T cells in the T-B border, IgM+ BMEM and IgM secreting LLPCs are also generated outside of the GC, in a GC-independent (GCi) manner. (iii) Short-lived antibody secreting cells (ASC) are generated early after activation to generate a rapid response against the pathogen. These short-lived ASC will undergo apoptosis and do not contribute to the generation of B cell memory. Bottom panel: Some protein antigens provide highly repetitive antigenic structures, which induce strong BCR crosslinking. Viral single-stranded RNA (ssRNA) together with other danger signals also activate toll-like receptors such as TLR7. These strong signals are enough to activate B cells in a T cell-independent (TI) manner and generate short-lived IgM secreting ASC and IgM+ BMEM. B1b and marginal zone (MZ) B cells are activated in a TI manner and provide a faster response against the pathogen.

more prominent within GCs during the recall response to a variant viral protein antigen rather than to the original inducing antigen when sequentially immunizing mice with variant Dengue envelope proteins with 63% amino acid identity (100). However, when using HAs from more closely related influenza viruses, with ∼82% sequence identity, the GC response was dominated by highly mutated memory B cells, which led to a worsened antibody response as compared to the primary encounters, even in the presence of an adjuvant (101). In the elderly, a poor adaptive capacity of B cells toward the drifted influenza epitopes has also been demonstrated. This resulted in the expansion of B cell memory targeting mostly conserved but less potent epitopes (102). In contrast, memory B cell expansion after H3N2 infection reflected imprinting toward strains encountered early in life but also adaptation to the infecting virus (103). These studies suggest that a certain degree of an antigenic difference is needed to induce a protective secondary antibody response by stimulating broadly cross-reactive low-affinity IgM+ memory B cells. High-dose and adjuvanted vaccines may improve VE when influenza vaccines strains are antigenically-different. The propensity for IgM+ memory B cells to dominate recall GC responses may be further determined by pre-existing antibodies that may outcompete the BCRs from low affinity naïve and IgM+ memory B cells, but not high affinity IgG+ memory B cells, for antigen (52, 96, 104).

In consideration of the potential for influenza Ab responses to become focused on epitopes present in successive vaccine strains to the detriment of recognizing future variants, it seems appropriate to think in terms of future vaccines that maintain plasticity and heterogeneity within the B cell response. For example, vaccination strategies that recall IgM+ memory B cells with less-mutated BCR repertoires, while also inducing naïve populations together with cognate Tfh cell memory to facilitate memory GC formation (98), may tend to skew the overall response toward the generation of more cross-reactive Abs against variant epitopes.

#### T Cell-Independent B Cell Responses Against Influenza

In contrast to TD Ags, which are generally proteins that cannot induce cross-linking of multiple BCRs, TI Ags are generally multivalent polysaccharides or other molecules that contain a repetitive array of antigenic epitopes that have that BCR-polymerization propensity. This paradigm is, however, challenged by the finding that high doses of a monomeric protein Ag can also elicit an exclusive TI B cell response (105, 106). In mouse experiments, both TD and TI B cells give rise to shortlived plasma cells and memory B cells (107–109) and contribute not only to resolving primary influenza virus infection, but also to more effective control of virus replication and symptoms after secondary challenge (110). The recall capacity of TI memory B cells is largely a result of Ag driven clonal expansion, however, like other memory B cells, TI memory B cells are able to respond more readily to Ag than their naïve counterparts.

The capacity of inactivated whole (vs. split) virion vaccines to induce superior influenza virus-specific antibody responses (111–113) may in part be due to the greater induction of TI B cell responses (114). Notably, when TI B cell responses were induced Ab affinity and neutralizing activity was enhanced. The ability of inactivated whole virions to induce TI B cell responses is linked to the presence of single-stranded RNAs that activate B cells via a TLR7-dependent mechanism (114), hence TLR7/8 agonists should be considered as potential adjuvants for seasonal influenza vaccines.

#### Importance of Location for Influenza-Specific Memory B Cells

Unlike LLPCs, memory B cells persist as tissue-resident or circulating among the SLO (115). Memory B cells resulting from a local infection also localize in the affected organs. This occurs following influenza virus infection when influenzaspecific memory B cells can be found, not only in lymphoid organs, but also in the lungs. Moreover, memory B cells are also differentially distributed among the lymphoid tissues, indicating that trafficking is influenced by local tissue factors (116, 117). After influenza re-exposure, lung-resident memory B cells differentiate into plasmablasts, providing IgG and IgA in situ that quickly neutralizes the virus (117, 118). In general, IgA+ memory B cells seem to localize preferentially to the blood and to tissue sites of pathology, while IgG+ memory B cells are broadly distributed among tissues that may, or may not, be directly involved in the disease process (116, 117). B cell memory and secreted IgA located in the lungs are essential to provide a quick and effective response against influenza viruses upon exposure, yet current influenza vaccines fail to strongly boost IgA responses (119). Antigen reaching the mucosa of the lung is required to potentially induce stronger IgA responses and for the generation of lung-resident memory B cells, which establish early after infection. The varied location of memory B cells according to their isotype, together with the fact that different environments drive B cell class-switching to a specific isotype, are of particular interest for vaccine design, particularly where (as in influenza) mucosal surfaces are the primary site of infection.

#### T Follicular Helper Cell Memory: Recent Advances in Influenza Vaccination

When the GC contracts, the GC Tfh cells exit and develop into resting memory Tfh cells with a less polarized Tfh phenotype (120–125). Tfh cells with a resting memory phenotype both recirculate in blood and can be found in BM, spleen, and lymph nodes (126–128). Circulating Tfh (cTfh) cells are the most accessible subset in humans. Of increasing research interest, cTfh cells are heterogeneous and can be classified into different subsets based on surface marker expression. Resting cTfh cells express CCR7, which differentiates them from their GC counterparts. When cTfh cells become stimulated, they downregulate CCR7 to traffic to the GC (129). Three different subsets of cTfh cells can be distinguished according to the surface expression of the chemokine receptors CXCR3 and CCR6, which are involved in inflammatory-homing and epithelial and mucosal site-homing, respectively (130, 131). The Tfh1 cells are CXCR3+ CCR6−, express the T-bet transcription factor and secrete the Th1 cytokine IFNγ. Conversely, the CXCR3−CCR6− Tfh2 set expresses the transcription factor GATA3 and produces the Th2 cytokines IL-4, IL-5, and IL-13. Then the Tfh17 cells CXCR3−CCR6+ cells express the transcription factor RORγT and secrete the Th17 cytokines IL-17A and IL-22 (132).

An overall consensus on the functional implications of the different Tfh subsets regarding B cell help is yet to emerge. While the Tfh1 cells are thought not to be efficient B cell helpers, the opposite is true for the Tfh2 and Tfh17 populations (132, 133). However, human studies on the cTfh response following influenza vaccination demonstrate an increase of circulating, activated cTfh1 cells peaking on day 7 after vaccination that positively correlates with the generation of protective Ab responses and the presence of ASCs in blood (115, 134). In the context of influenza immunization, when culturing human cTfh1 cells isolated at day 7 after priming with either naïve or memory B cells, the cTfh1 cells stimulate memory B cell differentiation into plasmablasts, while naïve B cells remain resting. Yet, naïve B cells cultured with Tfh2 and Tfh17 cells can differentiate into plasmablasts (134). Because Tfh cells are essential to induce a proper B cell response and we speculate that naïve B cells are not being sufficiently stimulated due to epitope masking by pre-existing Abs and memory B cells, it could be possible that mainly Tfh1 cells are stimulated after influenza vaccination at the expense of Tfh2 and Tfh17.

# Anti-viral CD8+ T Cell Responses

Seasonal influenza vaccines are designed to elicit an Ab response. However, the natural influenza virus infection additionally elicits cellular immunity (CD8+ T cells, CD4+ T cells, MAIT cells, NK cells) to eliminate the infection. Because influenza viruses are under constant selective pressure, the long-term protective value of any vaccine that targets a specific HA and/or NA will inevitably be compromised with time, immune CD8+ T cells are critical for recovery and provide some protection against severe influenza disease, including that resulting from infection with a previously unencountered avian strain. This likely reflects that influenza-specific CD8+ T cells tend to recognize HLAbound peptides derived from more conserved, internal virus proteins. The question is whether vaccines that promote such CD8+ T cell memory can, when combined with the classical products that induce virus-specific Ig response, provide better protection against, in particular, a newly invasive pandemic strain. An overview comparison between B and T cell responses after influenza virus drift and shift and how they complement each other is shown in **Table 1**.

Adaptive T cell immunity is mediated primarily by T cells, expressing the CD4 or CD8 co-receptors, respectively. During influenza virus infection, viral proteins are degraded by the proteasome and processed into smaller peptide fragments. These fragments are bound to MHC molecules and carried to the cell surface for presentation. These peptide/MHC complexes (pMHC) are recognized by clonally expressed TCRs on CD4+ or CD8+ T cells, leading to their activation and recruitment into the virus-specific immune response. The CD8+ cytotoxic T lymphocytes act as sentinels, recognizing and killing virusinfected targets, an essential step for virus clearance. Following activation, CD8+ T cells also secrete anti-viral cytokines (especially IL-2, IFN-γ, and TNF-α) which further recruit innate and adaptive immune cells into sites of influenza virus-induced pathology and induce anti-viral responses in infected cells (141, 142). When it comes to CTL killing, the secretion of perforin, granzymes and FAS ligand can all be involved in the process of inducing the apoptosis of virus-infected cells (143, 144). Additionally, the expression of TRAIL on CTLs can lead to the elimination of influenza virus infected cells, with a resultant decrease in mortality (145).

#### T Cell Fate: to Die or Become Memory

Formation of memory CD8+ T cells is essential for the protection against re-encountered pathogens. Our understanding of key factors determining the fate of CD8+ T cells during influenza is still limited but crucial for the development of a CD8+ T cell activating vaccine. During differentiation from naïve to effector, to memory status, CD8+ T cells transiently express cell surface molecules that are considered to be predictive of cellular fate and function. Surface expression of IL-7R and KLGR1 on effector CD8+ T cells can, at least in some situations, differentiate between CD8+ T cells designated as memory precursor effector cells and short-lived effector cells (146). Compared to the IL-7RloKLGR1hi set, CD8+ T cells expressing high levels of IL-7R and low levels of KLGR1 are 10-fold more likely to survive (147) in mice infected with lymphocytic choriomeningitis virus (LCMV). However, it should be noted that these profiles may not be exclusive, as KLRG1+ CD8+ T cells are detectable after LCMV infection is cleared (148), and the survival value associated with the IL-7RhiKLGR1lo set for LCMV is less obvious for influenza virus infection (149). Additionally, the discovery of other early markers of memory formation during Listeria monocytogenes and vesicular stomatitis virus infection, including expression of ID3 transcription factor (150) and IL-2Rα cytokine receptor, showed that CD8+ T cell memory generation is certainly multi-factorial (151, 152). Identifying markers of successful memory formation is crucial for evaluation of novel influenza vaccine responses and should be considered in future influenza vaccine studies. More recently, high-throughput sequencing is facilitating the emergence of a broader picture for CD8+ T cell differentiation. Single-cell RNAseq of CD8+ T cells at the acute phase of LCMV infection indicates that there may be two distinct populations of antigen-induced CD8+ T cells that share genes either with "terminal effector" or "memory" cells (153). Compared to naïve CD8+ T cells, the "terminal effector-like" set can be shown to have upregulated more than 900 different genes, while the "memory-like" cells only upregulated 27 genes (153). This suggests that the differentiation of "terminal effector" CD8+ T cells mandates the upregulation of hundreds of genes involved in both clonal expansion and the mediation of a spectrum of effector functions, while the establishment of CD8+ T cell memory requires only the involvement of a few key genes to maintain lymphocyte quiescence. Although the exact factors mediating distinct CD8+ T cell fates during early division following viral infection are still in the process of elucidation, experiments with TCR-transgenic mouse models indicate that TCR signaling strength (154), as reflected in IL-2R, IFN-γR, and mTOR levels during mitosis and asymmetrical division (155–157) is key to the generation of anti-viral CD8+ T cell memory. This is an exciting area of research that should, as it unfolds, give a much better understanding of both the molecular basis of CTL memory formation, and provide key measurement parameters that will allow us to skew early vaccine responses so that they provide optimal memory that gives long-lasting protection when recalled by further pathogen challenge.

#### Importance of Generating Long-Term T Cell Memory

As mentioned above, memory CD8+ T cells are important for eliciting long-term, broadly cross-reactive immunity to influenza viruses, and are thought to mediate the protective function mainly via the killing of virus-infected targets (158). Virus-specific CD8+ effector T cells also produce proinflammatory cytokines, and the breadth of cytokine production (termed polyfunctionality) often correlates with efficient protection against pathogens, including influenza viruses (159). Polyfunctional memory CD8+ T cells (producing IFN-γ, TNF, IL-2, and MIP-1β) (160) are thought to operate via augmented cytolytic activity via dual IFN-γ/TNF expression (161), IL-2-mediated enhancement of CD8+ T cell memory function (162) and increased IFN-γ secretion on a per cell basis (163). One example of the protective capacity of these polyfunctional memory CD8+ T cells is the induction of longlasting memory CD8+ T cells against variola (smallpox) virus induced by the Vaccinia vaccine Ankara (164). When CD8+ T cells were primed with influenza virus nucleoprotein (NP) expressed by either a recombinant Vaccinia virus or in Listeria monocytogenes, the more polyfunctional NP-specific CD8+ T

#### TABLE 1 | The clinical outcome and the B and T cell memory responses after exposure to influenza viruses are summarized below.

cells were generated following Vaccinia virus exposure. Mice vaccinated with the Vaccinia virus showed also a greater level of protection against a normally lethal IAV challenge compared to the Listeria monocytogenes vaccine group counterparts (165). This indicates, that not only the quantity of memory CD8+ T cells is critical for the protection but also their quality. Insights into key factors inducing these polyfunctional CD8+ T cells could improve a T cell-based vaccine therefore vastly.

Memory CD8+ T cells can be divided conceptually into central and effector T cell memory sets, based on their expression profiles for the CD62L and CCR7 surface proteins (166) that are known to affect cell localization and function (167). The CD62LhiCCR7hi "central memory" CD8+ T cells (TCM) can be found in the spleen, blood and lymph nodes, and display superior functions compared to their CD62LloCCR7lo effector memory CD8+ T cell (TEM) counterparts, mainly in terms of their proliferative capacity and IL-2 production profiles (168). In addition, a highly specialized population of tissue-resident (TRM) memory CD8+ T cells expressing CD103+CD69+ can persist in sites of pathology subsequent to virus clearance (169). Following the secondary challenge, CD103+CD69+ TRM set is able to expand and secrete cytokines, including IFN-γ and TNF, as well as generate more polyfunctional progeny (69% of cells capable of secreting three cytokines), when compared to CD103−CD69+ (21%) and CD103−CD69− (16%) parent subsets (160, 169). In the context of influenza, persistence of influenza-specific CD8+ TRMs correlates strongly with protection when mice are challenged with a serologically distinct IAV that shares common internal proteins (170). The TRM population develops from precursors lacking KLRG1 (171, 172) and further studies on T cell receptor (TCR) repertoires suggest that they arise from the same naïve pool as TCM set (173). TRM generation is largely regulated by a series of transcription factors (174), such as Runx3 which is crucial for TRM establishment across a range of tissues (175), and Bach2 which is recognized to restrain the terminal differentiation of effector T cells and help with formation of longterm memory T cells (176). The differentiated TRM phenotype is associated with changes in key transcription factors, including downregulation of Kruppel-like factor 2 (KLF2), TCF1 (177), T-bet, and Eomes (178, 179) as well as upregulation of Hobit, Blimp1 (177) and AhR (180), Nur77 (181), and Notch (182), required for the maintenance of TRMs. While the previously named transcription factors are universal hallmarks of TRM formation, TRM heterogeneity among cells generated at different tissue sites suggest that microenvironmental cues are important for site-specific TRM differentiation. Indeed, generation of the lung TRM set is influenced by transforming growth factor β (TGF-β) along with the presence of IFN-γ-secreting CD4+ T cells following influenza virus infection (183, 184). While the generation of influenza-specific TRMs has recently been shown to be vital for robust protection, unlike TRMs generated within the skin or gut (185–187), lung-resident TRMs do not offer long-term protection, rather they require a constant supply of circulating TEMs cells to replenish the niche over time (188) (summarized in **Figure 2**). In humans, influenza-specific lung-resident TRM cells show a high degree of TCR-sharing with other influenzaspecific lung TEM cells, suggesting that both memory cell subsets originate from the same precursors (160). Our understanding of the protective role of memory CD8+ T cells in influenza virus infection also comes from experiments with a C57BL/6 mouse model lacking antibodies, where increased numbers of influenza-specific memory CD8+ T cells and TRM cells led to markedly reduced influenza-induced morbidity (189). Similarly, primary vaccination with a single-cycle, non-replicative H3N2 IAV induced CD8+ T cells capable of protecting against a heterologous (H1N1) lethal challenge (190), an effect that was diminished for mice that had been depleted of CD8+ T cells after vaccination. These studies highlight the potential of longterm memory CD8+ T cells protecting against severe influenza virus infections. A potential that is not harnessed in the current vaccine strategy.

#### CD8+ T Cells Recognize Highly Conserved Influenza Epitopes

CD8+ T cells can confer broad cross-protection across different seasonal, pandemic and avian influenza IAV strains due to their ability to recognize relatively conserved viral peptides derived from internal influenza components (NP, M1 and PB1, PB2). The best defined human CD8+ T cell influenza epitope is the immunodominant M158−66 peptide bound to the HLA-A∗ 02:01 molecule (191–193). This peptide is highly conserved within different influenza A subtypes spanning 100+ years (136), including the 1918 and 2009 pandemic H1N1 strains as well as highly pathogenic H5N1 avian viruses (194). Analysis of immunogenic peptide profiles for the avian H7N9 influenza virus established that it shared six universal CD8+ T cell epitopes conserved at ∼100% prevalence in human influenza A viruses circulating since the catastrophic Spanish 1918 influenza. These universal human influenza-specific CD8+ T cells epitopes include HLA-A∗ 02:01/M158−66, HLA-A∗ 03:01/NP265−273, HLA-B∗ 08:01/NP225−233, HLA-B∗ 18:01/NP219−226, HLA-B ∗ 27:05/NP383−391 (although mutants were found in some H3N2 strains) and HLA-B∗ 57:01/NP199−207 (136). The population coverage by the universal HLAs varies greatly across ethnicities. Fifty-six percent of Caucasians displaying at least one universal HLA, while such coverage reached only 16% in the Alaskan and Australian Indigenous populations (136), highlighting the vulnerability of Indigenous populations toward newly-emerged influenza viruses. Additionally, our recent studies found broadly cross-reactive CD8+ T cell responses directed toward the HLA-B37-restricted NP338 epitope across IAVs (195), and excitingly, for the HLA-A ∗ 02:01-restricted PB1-derived epitope across influenza A, B and C viruses (196). The latter introduces a new paradigm

FIGURE 2 | CD8+ T cell memory formation. Naïve CD8+ T cells become activated by recognition of viral peptides presented in the context of MHC-I molecules on the surface of virally-infected APCs. Activated CD8+ T cells divide and differentiate into effector CD8+ T cells, which kill virus-infected cells and secrete cytokines to induce an anti-viral milieu. After viral clearance, mainly KLRG1lo, ID3+, IL2Rα +, and CD62Lhi CD8+ T cells develop into CD8+ memory T cells, while the remaining ∼90–95% of CD8+ T cells undergo apoptosis. Memory formation can be augmented by innate-like T cells (iNKT and MAIT cells). Memory CD8+ T cells are divided based on surface marker expression, known to impact their localization. While TCM and TEM can be found in blood and tissues, TRM reside at the site of infection where they can rapidly respond towards a secondary infection. TCM can be also found in lymph nodes and display higher proliferative capacity and IL-2 production compared to their TEM counterparts.

whereby CD8+ T cells can potentially confer a measure of previously unrecognized cross-reactivity across all human influenza A, B and C viruses, a key finding for the design of universal vaccines.

Influenza-induced morbidity and mortality can correlate with the expression of certain HLAs, including HLA-A∗ 24:02, A∗ 68:01 or B∗ 39:01 alleles, as shown during the 2009 H1N1 pandemic (197). Analysis of peptide scores demonstrated that HLA-A ∗ 24:02 is more likely to bind variable (rather than conserved) viral regions (197). Similarly, we have previously shown that some HLA alleles, including HLA-A∗ 24:02 and A∗ 68:01, are less able to elicit robust immune responses toward the highly conserved NP and M1 peptides (136). Both HLA-A∗ 24:02 and A∗ 68:01, in particular, are found at higher frequencies for Indigenous populations world-wide (136, 197), which may explain the disproportionate impact of pandemic influenza viruses on Indigenous peoples during both (otherwise mild) 2009 pH1N1 pandemic and 1918–1919 (H1N1) Spanish "flu catastrophe" (198–202).

Thus, given the broad potential for cross-protective capacity mediated by CD8+ T cells, along with more recent evidence that this effect may indeed be operating in nature to protect people, this aspect of immunity is of considerable interest in terms of developing improved influenza vaccines. However, it is important to note that designing peptide-based T cell vaccines that only cover the major HLA types would clearly be disadvantageous for Indigenous populations globally (203). Further research on CD8+ T cell epitopes found in high risk populations is therefore of highest importance to protect people of highest vulnerability.

#### CD8+ T Cells Can Confer Broad Cross-Protection for Heterologous IAV Strains

In the context of newly emerging influenza virus infections in people, correlative studies suggest that established CD8+ T cell memory confers cross-reactive immunity against severe influenza disease, as observed during the 2009 pandemic H1N1 (pH1N1) outbreak (139, 204). The high (∼70%) conservation of CD8+ and CD4+ T cell epitopes contributing to pre-existing memory may have been a significant factor in the generally mild outcomes of the 2009 H1N1 pandemic (138). Sridhar et al. showed that individuals with higher numbers of CD8+ T cells recognizing conserved influenza epitopes fared better following natural infection with the 2009 H1N1 virus (139). The importance of CD8+ T cell-mediated immunity was further highlighted in 2013 following the emergence of the novel avian H7N9 strain (205, 206), which killed ∼40% of the infected patients. In H7N9-infected individuals, rapid recovery from hospitalization was associated with the presence of significantly more IFN-γsecreting CD8+ T cells when compared to the situation for those who died (207) and recovered (206).

#### Development of CTL-Based Vaccines Lessons Learned From the Yellow Fever Vaccine

While the initial experience of IAV infection generally occurs in the first 6 years of life (208), our understanding of both the primary IAV-specific CD8+ CTL response and the transition to influenza-specific T cell memory is very limited for humans. Though one paper by Mbawuike et al. reported on primary infection in infants as early as 6 to 13 months of age (209), studies of such influenza exposures in infants are rare, and have not been performed using contemporary approaches for the analysis of T cell-mediated immunity. The closest we have for humans of any age when it comes to the formation of memory CD8+ T cells following first virus encounter is for the live-attenuated 17D yellow fever (YF) vaccine. As might be expected from a plethora of mouse experiments, recent YF vaccination studies showed that deuterium-labeled, epitope-specific CD8+ CTLs expanded initially following vaccination, before undergoing a contraction phase characteristic of CD8+ T cell memory. These vaccine-induced YF-specific memory CD8+ T cells persisted in the blood for at least 2 years after YF vaccination, with an average deuterium half-life decay rate of 493 days (210). A similar YF vaccination study in mice demonstrated that, after initial contraction, the long-lived CD8+ T cell memory pool remained consistent in size (211), indicating a potential advantage of a CD8+ T cell that would need fewer revaccinations compared to the annual recommendation necessary for the seasonal influenza vaccine. Unfortunately for influenza vaccination, the current IIV used in humans does not induce any CD8+ T cell responses that can be targeted for such a longevity analysis (115).

#### Vaccination Approaches to Induce Memory CD8+ T Cells

Different influenza vaccination approaches are currently being investigated in order to induce long-lasting cross-protective immunity. The only licensed vaccines capable of inducing CD8+ T cell immunity, such as the YF vaccine, use live-attenuated pathogens. These are not recommended for influenza "high-risk" groups such as pregnant women, immunosuppressed individuals and the elderly. Therefore, new vaccination strategies need to be developed if we are to protect such vulnerable people. Vogt et al. showed that changing the route of vaccine administration of a quadrivalent inactivated influenza vaccine from intramuscular (i.m.) to transcutaneous induces the expansion of vaccine component-reactive CD8+ T cells. Interestingly, the vaccine was also able to induce M158−66-specific responses in a HLA-A ∗ 02:01-positive donor, although this was only observed in one individual (212). Another approach currently in development is the Flu-v CD8+ T cell-activating vaccine (213) containing four 21–35 amino acid-long peptides from internal influenza proteins, which can potentially bind to multiple HLA allelic forms, including the highly prominent HLA-A∗ 02:01. This approach was protective for HLA-A2 transgenic mice and was also capable of inducing IFN-γ-expressing CD8+ T cells across all the participants (n = 15) in a phase 1b vaccine trial (213, 214). The Flu-v product showed that the vaccine reduces both the viral titer and the symptom score after H3N2 virus challenge in humans (215). However, due to the unknown HLA-restriction of the immunogenic epitopes, the HLA coverage of this vaccine is still to be determined. To circumvent the need for prior knowledge of HLA-restricted epitopes to be included in a universal T cell-based vaccine, particularly for less common HLA allelic variants, fulllength influenza proteins have been expressed in Vaccinia virus

Ankara vaccine vectors. Berthoud et al. showed that a viral vector encoding for the two internal proteins NP and M1 could induce some CD8+ T cell responses (216).

Overall, development of an effective, long-lasting, crossreactive influenza vaccine relies on an individuals' capacity to generate polyfunctional lung-resident CD8+ T cells. However, difficulties in identifying cross-reactive epitopes caused a bottleneck in the development of a universal influenza vaccine. Due to the propensity of IAV to trigger severe outbreaks with pandemic potential, murine models have thus far been developed to test the effectiveness of IAV vaccines based on conserved internal proteins (217–219). While mice immunized with these vaccines can elicit protective CD8+ T cell responses, the molecular mechanisms which govern formation of protective memory responses still require further validation in mice, and ultimately in humans.

# Innate and Bystander T Cell Activation During Influenza Virus Infection

In addition to the activation and proliferation of CD8+ T cells in a peptide-MHC dependent manner, T cells can also become activated via antigen-independent mechanisms, resulting in proliferation of polyclonal T cells (220). In an influenza mouse model, adoptive transfer of TCR-transgenic OT-I CD8+ T cells, which recognizes the ovalbumin peptide, into influenzainfected mice, showed that these OT-I cells can non-specifically expand in the lungs of influenza-infected mice. This suggests that CD8+ T cells can become activated independently of their TCRs during primary influenza virus infection (221). Similarly, highly activated CD38+HLA-DR+ CD8+ T cells, numerically greatly exceeding influenza-specific CD8+ T cell pools, were found in patients hospitalized with severe H7N9 disease (137), suggesting bystander activation of at least some CD38+HLA-DR+ CD8+ T cells. Despite the evidence that bystander CD8+ T cell activation occurs during influenza virus infection, the importance of these cells in terms of viral clearance and the induction of long-term memory is poorly understood. To date, the most solid evidence for the role of bystander activation has been observed in innate-like T cells. These cells, unlike conventional CD8+ T cells, recognize non-peptide antigens presented by orthologous MHC I-like molecules. They rapidly secrete cytokines following activation and can mediate some level of protection before adaptive immunity is sufficiently advanced (222). Recently, we demonstrated that mucosalassociated invariant T (MAIT) cells become activated during IAV infections in humans and mice (223, 224). These MAIT cells recognize riboflavin-derivative antigens produced by microbial pathogens (225), but can be variously activated by IL-12/IL-18 (224), IL-15, or type I interferons (226). Using a murine model, we showed that MAIT cells rapidly accumulate and become activated in the infected lung and contribute to protection against IAV infection (223). Similarly, invariant Natural Killer T (iNKT) cells, which recognize lipid antigens presented by CD1d, can protect against murine IAV (227–229). In addition, iNKT cells induced by inactivated influenza A virus vaccination in conjunction with alpha-galactosylceramide, an iNKT cell antigen, can boost influenza-specific memory CD8+ T cells and protective immunity in mice (230). The exact contribution of innate T cells vs. conventional CD8+ T cell-mediated immunity against influenza viruses is a subject of further investigation. These new insights help to understand the wider range of vaccine responses thus offering us opportunities to generate better strategies to fight against influenza.

# CONCLUDING REMARKS

Although current seasonal influenza vaccines can promote the induction of highly specific, long-term memory B cells that produce antibodies against the viral HA1 domain, these antibodies are generally unable to combat newly emerging influenza viruses, including novel pandemic stains and antibodyselected "seasonal" variants that have accumulated mutations in those epitopes surrounding the receptor binding pocket. Generation of high-affinity neutralizing Abs against conserved surface epitopes remains a constant challenge to provide longlasting and cross-protective B cell memory, and as such, more work is needed to better understand B cell responses against natural infection vs. vaccination, in order to design better B cell- or antibody-based universal vaccines. On the other hand, an influenza vaccine capable of stimulating CD8+ T cell responses would generate long-term T cell memory against conserved epitopes without the need for annual vaccination. In addition, a role for innate-like T cells in influenza protection is increasingly emerging, which could potentially be important both for the development of novel therapeutics and for boosting (or maintaining) long-term memory. As a consequence, substantial efforts are being made globally to exploit both innate and adaptive immune components for the development of novel influenza vaccines that induce longlasting B cell/antibody and/or cross-reactive T cell immune memory populations.

# AUTHOR CONTRIBUTIONS

MA and AF wrote the sections on influenza and humoral immunity. LH and XJ wrote the sections on cellular immunity. BC wrote the section on vaccine adjuvants. TN, KK, and PD wrote and modified the original manuscript, and the revised versions.

# FUNDING

KK is supported by the National Health and Medical Research Council (NHMRC) Program Grant (ID 1071916) and the NHMRC Senior Research Fellowship (ID 1102792). AF is supported by the NHMRC Program Grant (ID 1103367). MA and LH are supported by the Melbourne International Research Scholarship (MIRS) and the Melbourne International Fee Remission Scholarship (MIFRS) from The University of Melbourne. XJ is supported by CSC-UoM joint scholarship from The University of Melbourne. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.

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lung: diversity, function, and antigen specificity. PLoS ONE. (2011) 6:16245. doi: 10.1371/journal.pone.0016245

cells underlie waning heterosubtypic immunity. Sci Immunol. (2017) 2:eaag2031. doi: 10.1126/sciimmunol.aag2031

inflammation and CD8+ T cell response during acute influenza A virus H3N2 pneumonia. J Immunol. (2011) 187:1515. doi: 10.4049/jimmunol. 1190034

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Auladell, Jia, Hensen, Chua, Fox, Nguyen, Doherty and Kedzierska. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Remembrance of Things Past: Long-Term B Cell Memory After Infection and Vaccination

Anna-Karin E. Palm† and Carole Henry\*

Section of Rheumatology, Department of Medicine, University of Chicago, Chicago, IL, United States

#### Edited by:

Michael Vajdy, EpitoGenesis, United States

#### Reviewed by:

Johannes Trück, University Children's Hospital Zurich, Switzerland Claude-Agnes Reynaud, Institut National de la Santé et de la Recherche Médicale (INSERM), France

#### \*Correspondence:

Carole Henry<EMAIL_ADDRESS>
#### †Present address:

Anna-Karin E. Palm, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

#### Specialty section:

This article was submitted to Immunological Memory, a section of the journal Frontiers in Immunology

Received: 14 June 2019 Accepted: 16 July 2019 Published: 31 July 2019

#### Citation:

Palm A-KE and Henry C (2019) Remembrance of Things Past: Long-Term B Cell Memory After Infection and Vaccination. Front. Immunol. 10:1787. doi: 10.3389/fimmu.2019.01787 The success of vaccines is dependent on the generation and maintenance of immunological memory. The immune system can remember previously encountered pathogens, and memory B and T cells are critical in secondary responses to infection. Studies in mice have helped to understand how different memory B cell populations are generated following antigen exposure and how affinity for the antigen is determinant to B cell fate. Additionally, such studies were fundamental in defining memory B cell niches and how B cells respond following subsequent exposure with the same antigen. On the other hand, human studies are essential to the development of better, newer vaccines but sometimes limited by the difficulty to access primary and secondary lymphoid organs. However, work using human influenza and HIV virus infection and/or immunization in particular has significantly advanced today's understanding of memory B cells. This review will focus on the generation, function, and longevity of B-cell mediated immunological memory (memory B cells and plasma cells) in response to infection and vaccination both in mice and in humans.

Keywords: B cell memory, vaccination, mouse vs. human, influenza virus, infection

#### INTRODUCTION

One of the hallmarks of our immune system is the ability to "remember" past exposure to pathogens. Such exposure can be from infection or vaccination, and by remembering we are, ideally, fully protected from infection upon future encounter with the same pathogen (1). Although humoral immunological memory is mediated in part by serum antibodies secreted by long-lived plasma cells (LLPCs), these cells are usually not described as memory B cells. Instead, memory B cells are defined as long-lived and quiescent cells that are poised to quickly respond to antigen upon recall (2–5).

Both memory B cells and antibody-secreting cells (ASCs) are the product of antigen activation and, most often, interaction with cognate T helper cells. They can be IgM+ or immunoglobulin class-switched, and display germline or affinity-matured antigen receptors (B cell receptors; BCRs) (2, 6–8). Although generation of memory B cells does require ligation of CD40 (9), an early burst of both memory B cells and ASCs can form independently of GCs, as well as in T-cell independent responses (10–16). However, T-cell independent memory responses are beyond the scope of this review and will therefore not be thoroughly discussed here.

**98**

The terminal differentiation of B cells into ASCs is governed by a gene-regulatory network and modified by environmental stimuli as reviewed in Nutt et al. (17). ASCs can be divided into short-lived ASCs, including short-lived plasma cells and plasmablasts, and LLPCs. Plasmablasts are considered a transient population and can be either precursors of plasma cells (short- and/or long-lived; mainly in mice) or terminally differentiated effector cells activated during ongoing immune responses (mainly in humans) (18–23). In mice, within 2–4 d after infection, plasmablasts are found in extrafollicular zones and differentiate into plasma cells that secrete large quantities of antibodies. This early humoral response of lower affinity usually lasts a few days (24). In contrast, activation and differentiation of B cells within GCs allow the generation of plasma cells of high affinity that will then migrate to the bone marrow, where they can survive for decades and provide long-term humoral protection (25). Such LLPCs are key to maintaining long-term humoral immunity after infection or vaccination. They persist in the absence of antigen for decades after the original exposure (26). Although they exist in multiple lymphoid organs, the bone marrow is the home of the majority of plasma cells in mice (27, 28).

Most of what we know about the generation of plasma cells and memory B cells comes from mechanistic studies in mice. Because of massive differences between mice and humans in terms of life span and cell populations/phenotypes, the biology of mouse and human B cells differs. It is therefore important to also look toward in vivo lessons we have learned from humans.

#### LESSONS FROM MOUSE STUDIES

#### The Plasma Cell vs. Memory B Cell Fate Decision

Following antigen activation with a T-dependent antigen, naïve B cells will interact with cognate T cells at the border between the Band T-cell zones in the secondary lymphoid organs (**Figure 1a**). Here, the activated B cells will proliferate and make their first fate decision: whether to differentiate into extrafollicular ASCs or germinal center (GC)-independent memory B cells, or to move deeper into the follicle to form a GC (**Figure 1b**). A similar choice must then later be made in the light zone (LZ) of the GC, further discussed below. Although the molecular mechanisms for this decision have been extensively studied they have still not been completely elucidated, especially for memory B cell generation. Several studies have addressed the possibility of a "master transcription factor" for memory B cell differentiation, similar to Bcl-6 for GC B cells and IRF-4/Blimp-1 for plasma cells (29, 30). Although Bach2, or specifically high expression of Bach2, in LZ GC B cells has been pointed out as a factor promoting differentiation to memory B cells, a transcription factor unique to memory B cells is yet to be found (31–37). As recently reviewed, ZBTB32, KLF2, ABF-1, and STAT5 have been associated with memory B cell generation, but further studies are needed to understand their role (38).

#### Affinity

There is general consensus in the field that initial affinity for the antigen influences which differentiation pathway will be chosen by an antigen-activated B cell. Newly activated B cells with a relatively high affinity for the antigen will differentiate into shortlived extra-follicular ASCs (39). This ensures that the first burst of secreted antibody has enough affinity for the antigen to opsonize it and form immune complexes that will be directly cleared by phagocytosis, activate complement, and/or be presented on follicular dendritic cells (FDCs), thereby driving affinity maturation in the GC (30, 40). Conversely, antigen-activated B cells of lower affinity typically develop into GC-independent memory B cells. These are most often unmutated and unswitched (IgM+), although class-switched GC-independent memory B cells have been described (13). The GC-independent memory B cells provide a means of retaining adaptability potential within the memory B cell pool, and these cells can either be recruited later in the same response or recalled upon secondary encounter with the antigen.

#### GC Responses

The third fate choice for antigen-activated B cells is to upregulate Bcl-6 and move deeper into the follicle and start a GC reaction [excellently reviewed in Victora and Nussenzweig (30), Mesin et al. (40)]. Briefly, the GC B cells will go through multiple rounds of division in the dark zone (DZ) of the GC, each time introducing mutations in their antigen receptor (B cell receptor; BCR). This process of somatic hypermutation (SHM) leads to affinity maturation and ensures that B cells will specialize their binding to a particular antigen. The mutated B cells will then move to the LZ, where the new BCR will be tested against the antigen presented on FDCs. The B cells that manage to form a BCR with high enough affinity will receive survival signals and either return to the DZ to go through another round of division and SHM, or exit the GC as a plasma cell or a memory B cell.

Similarly to extrafollicular fate decisions, BCR affinity to the antigen seems to play a role also in the GC (37, 41, 42). Highaffinity B cells can bind and endocytose more antigen, and consequently present more antigen-derived peptides on class II MHC (MHCII). This higher density of peptide:MHCII on high-affinity B cells gives them an advantage in competing for access to T-follicular helper (Tfh) cells (43–45). In addition, each interaction with a Tfh cell is prolonged and intensified due to a feed-forward loop depending on peptide:MHCII density and CD40:CD40L ligation (45). This enhanced CD40:CD40L interaction causes down-regulation of Bcl6 and turning on of IRF-4 in the GC B cells, allowing them to differentiate into Bcl6loCD69hi plasma cell precursors before exiting the GC as plasma cells (46) (**Figure 1c**). In addition, IL-21 secreted from Tfh cells is required for plasma cell differentiation (47), further demonstrating the importance of long and strong B:T interactions for this fate decision. A fraction of the plasma cells leaving the GC will home to the bone marrow, where their survival depends on a number of factors in the plasma cell niche (**Figure 1d**). This will be further discussed below.

Memory B cells, on the other hand, are generated from lowaffinity GC B cells in the LZ and will eventually enter the

help received by low-affinity LZ GC B cells, these will not be instructed to turn on either the plasma cell or the GC B cell transcriptional program. Instead, up-regulation of Bach2, CCR6, EBI2, Ephrin-B1, and IL-9R, together with down-regulation of Bcl-6 and S1PR2, promote differentiation to memory B cells (e). To maximize the

likelihood of secondary antigen encounter memory B cells will then position themselves strategically in secondary lymphoid organs, become tissue-resident at the site of infection, or patrol as recirculating cells (f).

circulation as patrolling cells or take up residence in lymphoid or target organs (**Figures 1e,f**). The observation that memory B cells consistently are of lower affinity and have fewer mutations than plasma cells indicate that the former are generated before affinity maturation has allowed for the production of high-affinity BCRs. Indeed, an extensive study shows that memory B cells are formed early in the response whereas LLPCs are a later product (15). This temporal discrepancy also fits well with the Bach2 dynamics in memory B cells. Bach2 is required for memory B cell differentiation and only early GC B cells express Bach2, with the expression starting to decline from day 10 (37). Moreover, these experiments show that T cell help, in the form of CD40:CD40L interaction, represses Bach2-expression in GC B cells in a dosedependent manner. Thus, B cells with higher affinity typically have a lower expression of Bach2 and are therefore predisposed to choose re-entry to the DZ or commitment to the plasma cell transcriptional program. Conversely, relatively weak T cell help, as would be the case for lower-affinity B cells, maintains a relatively high Bach2-expression in LZ B cells, thus favoring a memory B cell fate (37). It is not clear how Bach2 determines memory B cell fate, but it is believed to act as a suppressor of transcription, particularly of Prdm1 (encoding Blimp-1) and of pro-apoptotic factors such as Bim and Puma (37, 48–51). Thus, it seems likely that lack of strong signaling, and consequently lack of instructions to start the plasma cell or GC B cell transcriptional program forces activated B cells into memory fate. Interestingly, memory B cells and naïve B cells, which are both quiescent with persisting differentiation potential, have similar transcriptional profiles, with the important exception of memory B cells seemingly being hardwired for quick responses (31, 33, 34, 36, 52).

Selection of B cells with a relatively low affinity into the memory compartment early in the response thus ensures that a certain poly-reactivity is maintained within the memory B cell

pool. Indeed, preservation of germline, or close to germline, encoded BCRs in memory B cells provides the memory B cell pool with clones that are able to respond quickly while still maintaining a higher degree of flexibility in terms of antigen binding. This flexibility would be lost should only memory B cells with high-affinity mutated BCRs persist in the memory pool. This idea can be illustrated by the observation that around 10% of memory B cells recognize variant antigen better than wild type protein, thus allowing for breadth of protection in a way that LLPCs do not (53). Conversely, by choosing only the highestaffinity GC B cells for plasma cell fate, the quality of the secreted antibodies is ensured to be very high.

#### Immunoglobulin Isotype

Another proposed determinant factor of plasma cell vs. memory B cell differentiation is immunoglobulin isotype. B cells that have switched to IgG, IgE, or IgA are more prone to differentiate to plasma cells than memory B cells (54–58). Interestingly, a recent study showed that even when B cells are forced to switch to IgG1 independently of AID, thus uncoupling the effects of SHM and class-switch recombination (CSR), the switched GC B cells were predominantly differentiating into plasma cells (58). Moreover, transcriptional analysis of IgM+ and IgG1+ GC B cells in the LZ revealed altered signaling through Nur77 in the switched B cells, associated with increased expression of chemokines associated with exit from the GC into the plasma cell compartment (58). Together, these studies indicate that intrinsic properties of a non-IgM BCR, probably in their signaling capacity, influences the plasma cell vs. memory B cell fate decision.

#### Marking Memory B Cell Precursors

Studies aiming at defining memory B cell precursors in the GC have found differential expression of several markers on subsets of GC B cells in the LZ. One such marker is the chemokine receptor CCR6, which has been shown to be dispensable for the initial generation but required for correct positioning of memory B cells as well as for optimal recall responses (59, 60). These CCR6+ GC B cells are generally of lower affinity, and have a phenotype closely resembling that of memory B cells (e.g., upregulated EBI2 and S1PR1, and down-regulated S1PR2) (60). A recent study describes a population of Ephrin-B1highS1RP2low GC B cells as memory precursor cells in the LZ, positioned close to the edge of the GC (61). In addition, a study focused on plasma cell precursors in the GC LZ proposes that a fraction of GC B cells in the LZ presenting as Bcl6lowCD69low are memory B cell precursors (46).

Finally, IL-9R is expressed on memory B cells as well as on a subset of LZ GC B cells concluded to be memory B cell precursors (62, 63). In addition to Bach2-requirement, optimal memory B cell generation also needs Tfh-derived IL-9 (63), and signaling through IL-9R on memory B cells is required for their recall response (64). Taken together, memory B cell precursors may be found in the GC LZ and present as CCR6+S1PR2lowEphrin-B1highBcl6lowCD69lowIL-9R+. However, further studies are needed to fully elucidate whether this phenotype really corresponds to a committed memory B cell precursor.

# The Memory B-Cell Niche and Recall Responses

Upon re-exposure to an antigen the memory recall response will be faster, stronger, and more specific than a naïve response. Protective memory depends first on circulating antibodies secreted by LLPCs (**Figure 2a**). When these are not sufficient for immediate pathogen neutralization and elimination, memory B cells are recalled. It is therefore of vital functional importance that memory B cells are stationed at strategic sites where they can maximize their chance of encountering antigen (**Figure 2b**). The spleen, including the marginal zone, is a major reservoir for memory B cells in both mice and humans (14, 65–67), as is the subcapsular sinus (SCS) of lymph nodes (68). Both the splenic marginal zone and the lymph node SCS are abundant with CD169+ macrophages, which are specialized in presenting unprocessed antigen to B cells (69, 70). It has been demonstrated that both naïve and memory B cells interact with CD169+ macrophages in the SCS, and that upon antigen recall the memory B cells quickly form SCS proliferative foci (**Figure 2c**), or form new GCs (68). This was also seen in human lymph nodes. Interestingly, the largest output from the SCS proliferative foci is short-lived plasma cells (ASCs), whereas the new GC is a site for further affinity maturation and CSR with very stringent quality controls that limit plasma cell differentiation (42). Importantly, both the SCS proliferative foci and the GC also foster memory B cells that may participate in another recall response or be recruited later in the same response. In addition to the spleen and lymph nodes, memory B cells are found in the bone marrow, Peyers' patches, gingiva, mucosal epithelium of tonsils, the lamina propria of the gastro-intestinal tract, and in the circulation (67, 71–76). It has not been convincingly demonstrated that the bone marrow, or any other tissue (apart from the spleen and the lymph nodes) contains functional memory B cells or if these memory B cells simply recirculate from the blood to the tissues. These are all anatomical sites where antigen may breach the barriers or be carried to via the circulation, and the memory B cells located here act as sentinels should pre-existing antibodies not provide adequate protection.

Importantly, memory B cells can also seed sites of infection, where they are maintained as tissue-resident memory B cells (77–79). Here they are quickly activated after pathogen invasion without the need for antigen transportation to draining lymph nodes, thus shortening the time for plasma cell differentiation and antibody production on secondary exposure. Interestingly, in the case of influenza virus infection, broadly reactive memory B cells are enriched in the lung-resident pool, thus conferring quick and cross-reactive protection at the site of infection (80).

Upon re-exposure to antigen, memory B cells can quickly proliferate and differentiate into plasma cells. Alternatively, they will re-enter GCs for another round of affinity maturation and CSR. This decision depends on BCR affinity and isotype in addition to differential expression of CD80 and PD-L2 (**Figures 2d–f**). These surface markers denote functionally different memory B cells independent of immunoglobulin isotype (2, 4, 7, 8, 65). Importantly, the heterogeneity of the memory

B cell compartment allows for a functional breadth of memory recall responses.

Unswitched (i.e., IgM+) memory B cells are often derived from GC-independent or very early GC responses. They frequently do not express CD80 and/or PD-L2, and carry few, if any, mutations (7, 65). The IgM+ memory B cell pool thus keeps a breadth of reactivity similar to that of naïve B cells but with the advantage of being able to rapidly respond to antigen (31, 33, 34, 36, 52). This breadth is particularly important for mounting rapid recall responses to variant antigens, such as influenza virus. On the other hand, recalled IgG+ memory B cells tend to rapidly differentiate into plasma cells without re-entering a GC (4). This is comparable to the fate chosen by switched B cells in the primary GC response (54–58). However, these observations may not be exclusively dependent on immunoglobulin isotype. Indeed, when further dissecting the memory B cell compartment, it becomes apparent that CD80−PD-L2− IgM+ memory B cells preferentially enter GCs upon recall, whereas those expressing CD80 and/or PD-L2 typically generate rapid IgM+ and IgG+ plasma cell responses (4, 7, 8, 74, 81). Similarly, IgG+ memory B cells single-positive for CD80 or PD-L2 can differentiate to ASCs while retaining the capacity to seed GCs, whereas double-positive IgG+ memory B cells only generate ASCs (8). These findings are further supported by studies demonstrating that IgG+ and IgA+ memory cells can engage in new GC reactions (5, 75).

# LESSONS FROM HUMAN STUDIES

#### Human Plasmablasts

In humans, most studies consider plasmablasts as blood shortlived ASCs generated in acute B cell responses to infection or vaccination that transiently contribute to the serum antibody. In a secondary systemic immune response to a protein antigen such as tetanus toxoid or an inactivated influenza virus vaccine, antigen-specific IgG-secreting plasmablasts with somatically mutated VH gene rearrangements are generated from memory B cells (20, 82). It is also the case following influenza, Ebola, or Dengue virus infection (22, 83–85). It remains an open debate whether human plasmablasts are precursors of and how many do become LLPCs. Evidence suggests that once the infection is cleared, the majority of ASCs undergo apoptosis, while a small proportion may go on to further differentiate into LLPCs (86). The heterogeneity seen in human ASCs from tonsil, blood, and bone marrow reveals stages of increasing maturity, and local profiles of adhesion molecule expression suggest a multi-step model for plasma cell differentiation (82, 87). In human blood when plasmablasts appear between days 6 and 8 after vaccination, they are migratory and attracted by CXCL12 and could migrate to tissues, such as the bone marrow (88, 89).

Plasmablasts have also been described as a "steady state" population where the majority express IgA. They express CCR10 and the adhesion molecule β7 integrin and they are attracted by CXCL12 suggesting that they come from mucosal immune reactions and can return to mucosal tissue. Approximately 40% of LLPCs in human bone marrow are IgA+, nonmigratory, and express β7 integrin and CCR10, suggesting a substantial contribution of mucosal plasma cells to bone marrow resident LLPCs (90). After tetanus vaccination, IgG+CD62L+β7 integrin− dividing, vaccine-specific, and migratory plasmablasts appear in the blood, as do non-dividing, non-migratory, CD62L− plasma cells of different specificities (90).

A recent study identified survival factors from the bone marrow niche that favors maturation of human blood ASCs to LLPCs in vitro (91). IL-6 and two members of the tumor necrosis factor (TNF) superfamily: BAFF (B-cell activating factor of the TNF family; also known as BLyS in humans) and APRIL (a proliferation-inducing ligand) are known to be important survival signals (92), as well as is CXCL12 (93). Additional factors secreted by the bone marrow niche such as fibronectin and YWHAZ are important for LLPC maturation (91).

#### LLPCs

#### Migration to and From the Bone Marrow

Human LLPCs freshly isolated from the bone marrow have high expression of the chemokine receptors CXCR4 and CXCR6 and responsiveness in in vitro migration assays to the chemokines CXCL12 and CXCL16. The chemokine CCL28 has also been shown to attract human bone marrow plasma cells in vitro (94). Two interesting populations have been observed in the blood of tetanus toxoid immunized individuals: a population of migratory plasmablasts expressing CXCR3 and CXCR4, and a population resembling mature plasma cells of the bone marrow. These findings suggest that these cells are likely to be resident LLPCs mobilized from their survival niches in the bone marrow, in competition with newly generated plasmablasts (88).

#### In the Bone Marrow

Mesenchymal stromal cells (MSC) in the human bone marrow microenvironment provide factors that support LLPC survival (95–97). Cytokines of the TNF superfamily (BAFF, APRIL and TNF-α), IL-6 family, CD80/CD86, CD44 binding to hyaluronic acid, and VLA-4 binding to VCAM-1/fibronectin promote survival of plasma cells. CXCL12 promotes entry of cells to the bone marrow as well as plasma cell survival (86). BAFF seems to be important for human plasmablast differentiation whereas APRIL is the key to long-term survival in the bone marrow (98). An interesting study demonstrated that extracellular vesicles from bone marrow-derived MSCs support ex vivo survival of human ASCs (99).

In humans, the bone marrow contains both CD19+ and CD19− LLPCs (26). The majority of CD19− LLPCs are actually found in the bone marrow, compared to the blood, spleen and tonsils. Interestingly, CD19− LLPCs are enriched in IgG+ cells and carry fewer VH mutations compared to CD19+ LLPCs. Only CD19− LLPCs resist to mobilization into the blood following immunization, and are resistant to depletion by Rituximab. In addition, CD19− LLPCs were not found in the bone marrow of 5–7 months old infants while CD19+ LLPCs were present. This study suggests a multi-layer model of LLPCs in the human bone marrow with CD19+ LLPCs being a dynamic component and CD19− a more static component permitting both adaptation and stability of humoral protection (100). A more recent study of the same populations but this time in response to influenza virus vaccination suggests that newly generated ASCs can acquire a mature plasma cell phenotype that is accompanied by loss of CD19 expression at an early stage of differentiation, and that aging is not an obligate requirement for a CD19− state to be established (101). Finally both CD19+ and CD19− vaccinia-specific LLPCs were detected in the BM more than 35 years after the eradication of smallpox, suggesting that the LLPC pool may be maintained by a process in which vaccinia-specific B cells differentiate into LLPCs in the BM (26).

#### Outside of the Bone Marrow

Compared to the bone marrow niche, fibroblasts from the lymph nodes and the spleen have been poorly characterized in both mice and humans. A few studies have shown that stromal cells in the spleen and lymph nodes might promote plasma cell survival in vitro (102, 103). Recently, a new subset of fibroblasts (FRCs for fibroblastic reticular cells) in the lymph nodes have been described both in mice and humans as the main cell type in contact with plasma cells to guide them in their migration (104).

## Mucosal Plasma Cells

Plasma cells are very abundant in mucosal tissues. They are located both in the connective tissue (lamina propria) and in lymphoid organs such as the tonsils in the oral cavity and Peyer's patches in the gut. The majority of these plasma cells secrete IgA antibodies, and humans also have a substantial IgM+ plasma cell population in the mucosa (105). B cells in the respiratory tract and IgA responses in the gastrointestinal tract in have been nicely reviewed in Kato et al. (106) and Bunker and Bendelac (107), respectively, and are both beyond the scope of this review.

#### Human Memory B Cells

A great variety of B cell subsets have been identified in the tonsil, spleen, and peripheral blood and represent different stages of development of a naive B cell into a memory B cell. In the human tonsil, at least five distinct subpopulations of mature human B cells (Bm1–Bm5) have

been identified. Concisely, naive B cells belong to the Bm1 and Bm2 subpopulations whereas fully differentiated memory B cells belong to the Bm5 subset (108–110). Interestingly IgG transcripts in the tonsil had accumulated twice as many mutations as the IgM transcripts suggesting that reentry of selected B cells in the GC to generate higher affinity BCRs is a possibility (109).

As we previously stated, memory B cells are mainly generated in the GCs in secondary lymphoid organs. After leaving the GCs, memory B cells either join the recirculating pool of lymphocytes, or home to antigen draining sites. Memory B cell niches outside of the blood have been described and memory B cells have been found in the bone marrow, the tonsil and the spleen (111). Additionally a population of tissue based memory B cells expressing Fc receptor-like 4 (FCRL4) instead of CD27 has been described (112, 113). In the blood and bone marrow, human memory B cells can be divided in three main populations: CD19+CD27+IgM+IgD+ (similar to marginal zone (MZ) B cells), CD19+CD27+IgM+IgD− (IgM-ONLY) and class-switched CD19+CD27+IgM− (IgG+ or IgA+) (114, 115). An in-depth flow cytometry analysis of human bone marrow and blood samples showed that compared to the blood, the bone marrow was enriched in both MZ and switched B-cells (116). In the spleen, two main phenotypically distinct B cell populations exist and localize to separate areas of the lymphoid tissue. Mantle zone B cells (IgDhighIgM+CD21+CD23+) are unmutated and believed to be naive B cells, whereas MZ B cells are IgD+IgMhighCD21highCD23± and exhibit somatic mutations (117–119). It has been demonstrated that CD148, as well as CD27, are markers for memory B cells present in the human spleen (120). More recently, a population of IgG+ memory B cells residing in the MZ of the spleen have been found and examined. IL-21 and BAFF have been demonstrated to be important for the differentiation of these IgG+ splenic memory B cells into plasma cells (121).

#### CD19+CD27+IgM+IgD+ (Also Called Human MZ B Cells)

The spleen is an important organ in the defense against encapsulated bacteria. A population of "IgM memory B cells" controlling Streptococcus pneumoniae is observed in the spleen (122). Additionally, the human peripheral B-cell compartment displays a large CD19+CD27+IgM+IgD+ memory B cell population, resembling the splenic MZ B cells. In fact, by CDR3 spectratyping and gene-expression profiling, it has been demonstrated that CD19+CD27+IgM+IgD+ memory B cells are circulating splenic MZ B cells. These memory B cells have a mutated BCR, provide a pre-diversified immune repertoire and are involved in T-independent responses (123). They can develop in the absence of a spleen, but splenectomy in older individuals dramatically reduces the number of blood MZ B cells (122, 124). Finally, when compared to switched memory B cells in children <2 year of age, CD27+IgM+IgD+ memory B cells in the spleen and blood do not display any signs of antigen-driven activation and expansion despite the many antigenic challenges experienced during childhood, suggesting a developmental diversification outside of T-dependent and T-independent responses (125).

#### CD19+CD27+IgM+IgD− (IgM-Only) and Class-Switched CD19+CD27+IgM

By tracking tetanus toxoid-specific memory B cells (CD3−CD19+CD20+CD27+) at steady state, it has been showed that the spleen is the largest reservoir of memory B cells followed by the tonsil. Bone marrow and blood memory B cells express surface IgG and IgA at similar frequencies, while the tonsil contained more IgA memory B cells compared to other locations. IgG+ memory B cells were enriched in the spleen and the tonsil compared to the bone marrow and the blood and IgM+IgD+ memory B cells were reduced in the tonsil compared to other locations. Interestingly, the absence of spleen and tonsils does not affect secondary responses to tetanus, suggesting an organ independent maintenance and reactivation for human memory B cells (111). Memory B cells that reside in lymphoid organs and recirculate after re-exposure to antigen are phenotypically the same and do not represent different stages of maturity. Additionally, it has been demonstrated that the human spleen is a major reservoir of long-lived vaccinia-specific memory B cells (66). Indeed, anti-smallpox IgG+ memory B cells were specifically enriched in the spleen, confirming that the spleen is a major reservoir for long-lived memory B cells.

Finally, high-throughput VH sequencing on paired blood and spleen samples revealed that IgM sequences from clones shared between the MZ and the memory IgG/IgA (switched) compartments displayed a mutation and repertoire profile of IgM-only and not of MZ B cells. Thus the "IgM-only" subset appears as the only subset showing precursor–product relationships with CD27+ switched memory B cells, indicating that they represent GC-derived IgM memory B cells and that IgM-only and MZ B cells constitute two distinct entities (126).

#### Human IgG and IgA Responses Induced by Infection and Vaccination

The route by which an antigen enters the body (systemic vs. mucosal) and the nature of the antigen are factors that direct the immune response class-switching patterns. Protein antigens usually trigger B cells receiving T-cell help while polysaccharide antigens induce CSR in the absence of T-cell help. Moreover, BAFF and APRIL have been shown to stimulate CSR to IgG and IgA in human B cells (127). Polysaccharide B cell responses to vaccination in humans have been reviewed in Mitchell et al. (23), while the kinetics of ASC responses to infection have been reviewed in Carter et al. (128).

#### IgG

Antibody responses to soluble protein antigens and membrane proteins primarily induce IgG1, but are accompanied with lower levels of the other subclasses. Viral infections in general lead to IgG antibodies of the IgG1 and IgG3 subclasses (129). On the other hand, antibody responses to bacterial capsular polysaccharide antigens is almost only restricted to IgG2 (130). IgG4 antibodies are often formed following repeated or longterm exposure to antigen in a non-infectious setting (131).

#### IgA

Homeostatic IgA responses employ a polyreactive repertoire to bind to a broad subset of microbiota species and tend to be of low affinity. In contrast, mucosal pathogens and vaccines elicit highaffinity, T-cell dependent antibody responses (107, 132). Mucosal IgA responses through a T-cell dependent reaction that place in mucosal lymphoid follicles, such as intestinal Peyers' patches and mesenteric lymph nodes (together called MALT for Mucosa-Associated Lymphoid Tissues) (132). Human IgA subtypes show distinct anatomical expression patterns, with monomeric IgA1 dominating in the serum and dimeric IgA2 in the gut (133).

Very few studies in humans have compared the induction of IgA and IgG secreting cells following various routes of immunization. An early study compared oral, intranasal and systemic influenza virus vaccines in healthy adults. Both systemic and intranasal immunizations induced mainly IgG+ influenzaspecific B cells in the blood after vaccination while the oral route induced IgA+ influenza-specific B cells in the blood. Additionally, oral and intranasal administration of antigeninduced IgA influenza-specific antibodies in external secretions (134). These results were confirmed later on by multiple studies reporting a bursting population of IgG+ antigenreactive plasmablasts in the blood after secondary tetanus toxoid vaccination (88), influenza virus vaccination or infection (20, 83, 135), as well as acute dengue virus infection (22). In addition, immunization of African green monkeys with a live-attenuated H5N1 influenza vaccine resulted in more serum IgG neutralizing antibodies than IgA (136).

A study employing Ad26/Env (HIV) vaccination in rhesus macaques demonstrated highly coordinated IgG and IgA responses in both peripheral blood and mucosal compartments (137). It remains unclear to this day how related IgG and IgA plasmablasts/plasma cells are and what the relationship between mucosal and systemic antibody responses looks like. While a study suggested that mucosal and systemic humoral immune responses are regulated independently of each other based on the observation that systemic vaccination does not seem to impact peripheral IgA+ plasmablast numbers (90, 138), another study revealed that in celiac disease patients, the same antigen-reactive B cell clones that give rise to gut plasma cells also contribute to the serum IgG and IgA antibody pool. However, serum IgA antibodies had a molecular composition (IgA1 vs. IgA2 and J chain level) distinct from that of IgA antibodies secreted in the gut, suggesting the involvement of different plasma cell populations (139). Finally, analysis of long-term transcriptional profile between blood IgG and IgA influenza-reactive plasmablasts as well as influenza-negative IgA plasmablasts did not reveal any specialization based on isotype. These data suggest that IgG and IgA vaccine–positive plasmablasts are largely similar, whereas IgA vaccine–negative plasmablasts appear to be transcriptionally distinct from antigeninduced peripheral blood plasmablasts (140).

#### Lessons From HIV

Significant efforts in the HIV field are focusing on the design of vaccines that would induce the generation of broadly neutralizing antibodies (bNAbs). Understanding the immunology behind

Palm and Henry Long-Term B Cell Memory

the development of antibody potency and breadth following immunization is crucial in this context, not only to the HIV community (141). The success of most vaccines relies on the generation of antibodies to provide protection against subsequent infection. As discussed earlier in this review, Tfh cells are critical for the production of high-affinity B cell clones in the GC and thus the generation of long term memory, i.e., memory B cells and LLPCs (142).

The feasibility of assessing GCs and Tfh responses from human lymph nodes has been limited, as GC B cells do not circulate in the blood, and lymph nodes are rarely sampled (143). Recently, fine needle aspirates of the draining lymph nodes were used to longitudinally sample GC B cells and GC Tfh cells in non-human primates. The lymph node fine needle aspiration technique has proven effective in terms of how many cells were recovered from the biopsy as well as in not disrupting the ongoing GC. The authors found that neutralizing antibodies in nonhuman primates correlate with GC B cell magnitude and Tfh help quality (144). They also found that GCs peak weeks after the initial immunization. This means that a classic immunization (one injection of antigen) is not optimal for "feeding" the peak GC response. Proteins that are not of extreme stability can be degraded, exposing epitopes that would normally be hidden or non-existent on a more native protein conformation. Slow immunogen release could improve the availability of intact antigen and epitopes of interest for the duration of the GC response (145).

Germline-targeting strategies aim to activate B cell precursors with potential interest for bNAbs generation, so that they will enter the GC, be selected and affinity matured and will generate memory B cells. Studying HIV-reactive B cell lineages to infer unmutated ancestral BCRs that represent what a naïve B cell would express is the key to a B-cell lineage vaccine strategy (146). A vaccination protocol based on B-cell lineage differs from classic protocols in the fact that they may prime with one immunogen and boost with another or with a sequence of several different immunogens (147–150).

It has been recently demonstrated that only immunogens above a certain affinity and in multimeric form are capable of inducing GCs dominated by B cells from a bNAb precursor starting with low precursor frequency (151). These B cells successfully competed in GCs, underwent somatic hypermutation and differentiated into memory B cells. Overall this study demonstrates that germline-targeting immunogens can overcome affinity, avidity, and inter-clonal GC competition challenges with high-affinity multimeric designs.

#### Lessons From Influenza

Plasmablasts have been extensively studied in humans, especially in the context of influenza vaccination and infection. Little is known about B cells that become activated but do not differentiate into plasmablasts. A subset of antigen-reactive B cells called ABCs for "Activated B Cells" has been described and was found to be transcriptionally distinct from the ASC population and committed to the memory lineage (152). ABCs and ASCs share hemagglutinin (HA)-reactive clones following influenza vaccination. Our laboratory also described a post-GC population of B cells that phenotypically resemble memory B cells but that have low expression of CD21 (classical memory B cells are CD19+CD27+CD21high). We demonstrated that the CD21low population was comprised of recent GC graduates that were refractory to GC reentry and seemed to be predisposed to differentiation into long-lived plasma cells (153). Although clonally related to memory B cells and plasmablasts, CD21low B cells form distinct clades within phylogenetic trees based on the accumulation of variable gene mutations. Another study demonstrated that HA-reactive CD21low B cells are enriched in the blood compared to the tissues while there was an enrichment of CD27+CD21highHA+ B cells in all tissues. Both CD21+ and CD21low populations were not maintained in the peripheral blood at 1 year post-vaccination (154).

Additionally, it is of great interest to understand how different vaccine compositions will affect the generation of memory B cells and LLPCs. Seasonal influenza vaccines exist as live-attenuated influenza virus (LAIV), which more closely resembles natural immunity after infection, or as inactivated vaccines. LAIV have been used mostly in children but do not induce strong systemic antibody responses in adults (155). The same was true for two different avian pandemic LAIV vaccines (H5N1, H7N9), although these vaccines elicited a long-term immune memory that was revealed after administration of a matched inactivated vaccine (156–158). To understand how LAIV vaccines can prime such a memory response, a detailed analysis of B cell responses in systemic and local lymphoid tissues in a non-human primate model was performed (136). Interestingly, the authors found that the LAIV vaccine induced robust GCs in the mediastinal (lungdraining) lymph node and that both HA-reactive plasmablasts and memory B cells were found in the mediastinal lymph nodes after immunization.

Finally, it is believed that adjuvants can modulate humoral responses and retain antigen at the site of injection.

#### REFERENCES

Most studies have been done with alum and it remains unknown how other adjuvants (such as AS03 and MF59) act on GCs and antigen release (159). In the context of influenza vaccines, adjuvanted vaccines administered in patients with impaired immune responses, such as infants and the elderly, were shown to be beneficial (160–162). Additionally a study showed that the adjuvant AS03 induced an increased activation of naïve B cells and an increased adaptability of recalled memory B cells, improving immunogenicity (163).

#### CONCLUSION

The generation of memory B cells and long-lived plasma cells is crucial to the long-term effectiveness of vaccines. Understanding how to induce these different populations and modulate their effects both in animal models and human is essential to the design of better vaccines. Thus, the design of new immunogens, how to release them, as well as the mechanisms of actions of various adjuvants are the future of vaccines protecting against challenging or emerging infectious diseases.

#### AUTHOR CONTRIBUTIONS

A-KP and CH contributed ideas and wrote the review. A-KP designed the figures.

#### ACKNOWLEDGMENTS

We thank Patrick C. Wilson and Christopher T. Stamper for critical discussion. CH is supported by the National Institute of Allergy and Infectious Disease, National Institutes of Health, Department of Health and Human Services, CEIRS contract HHSN272201400005C.

cell survival and immunoglobulin secretion. Nat Commun. (2018) 9:3698. doi: 10.1038/s41467-018-05853-7

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Palm and Henry. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Virus-Specific Secondary Plasma Cells Produce Elevated Levels of High-Avidity Antibodies but Are Functionally Short Lived

Caroline C. Krueger 1,2, Franziska Thoms 3 , Elsbeth Keller 1,2†, Monique Vogel 1,2 and Martin F. Bachmann1,2,4 \*

1 Department of Rheumatology, Immunology and Allergology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland, 2 Department for BioMedical Research, University of Bern, Bern, Switzerland, 3 Department of Dermatology, University Hospital Zurich, Schlieren, Switzerland, 4 Nuffield Department of Medicine, The Jenner Institute, The Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, United Kingdom

# Edited by:

Michael Vajdy, EpitoGenesis, United States

#### Reviewed by:

Ali Ellebedy, Washington University in St. Louis, United States Ramon Arens, Leiden University Medical Center, Netherlands

#### \*Correspondence: Martin F. Bachmann

<EMAIL_ADDRESS>
#### †Present address:

Elsbeth Keller, Neurocenter, Vetsuisse Fakultät Bern, Bern, Switzerland

#### Specialty section:

This article was submitted to Immunological Memory, a section of the journal Frontiers in Immunology

Received: 03 October 2018 Accepted: 19 July 2019 Published: 06 August 2019

#### Citation:

Krueger CC, Thoms F, Keller E, Vogel M and Bachmann MF (2019) Virus-Specific Secondary Plasma Cells Produce Elevated Levels of High-Avidity Antibodies but Are Functionally Short Lived. Front. Immunol. 10:1831. doi: 10.3389/fimmu.2019.01831 Most vaccines aim at inducing durable antibody responses and are designed to elicit strong B cell activation and plasma cell (PC) formation. Here we report characteristics of a recently described secondary PC population that rapidly originates from memory B cells (MBCs) upon challenge with virus-like particles (VLPs). Upon secondary antigen challenge, all VLP-specific MBCs proliferated and terminally differentiated to secondary PCs or died, as they could not undergo multiple rounds of re-stimulation. Secondary PCs lived in bone marrow and secondary lymphoid organs and exhibited increased production of antibodies with much higher avidity compared to primary PCs, supplying a swift wave of high avidity antibodies early after antigen recall. Unexpectedly, however, secondary PCs were functionally short-lived and most of them could not be retrieved in lymphoid organs and ceased to produce antibodies. Nevertheless, secondary PCs are an early source of high avidity antibodies and induction of long-lived MBCs with the capacity to rapidly differentiate to secondary PCs may therefore be an underestimated possibility to induce durable protection by vaccination.

Keywords: adaptive immunity, anti-viral immunity, memory B cells, secondary plasma cells, virus-like particles

# INTRODUCTION

B cells differentiate to antibody secreting plasma cells (PCs) upon activation by their cognate antigen (Ag) within and outside of B cell follicles. At an early stage of the primary immune response, antibody–forming cells (AFCs) derived from follicular or marginal zone (MZ) B cells are rather short-lived and survive for a few days only (1). Meanwhile, follicular B cells form GCs where MBCs and long-lived PCs are generated in a mostly T cell dependent fashion (2–5).

Activated B-lymphocytes are driven to the PC pathway by up-regulation of the transcription factors B lymphocyte maturation protein 1 (Blimp-1), Interferon regulating protein 4 (IRF 4), and X-box-binding protein 1 (XBP 1) (6–8). Differentiation of activated B cells into AFCs needs a harmonized change in the gene expression of these cells. Shi et al. delineated the transcriptional profile during this differentiation process (9). PCs are terminally differentiated and arrested in the G1 phase of the cell cycle being incapable of further growth or proliferation (10, 11). To be able to secrete large amounts of antibodies, PCs are committed to their protein synthesizing machinery and undergo major structural adaptations by increasing the size of the endoplasmic reticulum and Golgi apparatus (12). To cope with these changing conditions PCs induce the unfolded protein response as well as autophagy (13–15). These stressregulating processes are necessary for survival as PCs can secrete the tremendous amount of up to 10'000 antibodies per second (16). Sizeable amounts of antibodies that are rapidly available are required to neutralize microorganisms and prevent infection. Antibodies furthermore play a key role in immunity and promote the crosstalk between the innate and adaptive immune system. Besides classical neutralization of toxins and pathogens, they are able to opsonize microbes and infected cells for phagocytosis, enabling their elimination, and promote antigen presentation thereby regulating inflammation (17).

PCs are found in secondary lymphoid organs and the bone marrow (BM) where they can survive for days, months, or even years. There is an ongoing debate whether long-term antibody responses are a result of persisting antigen leading to re-stimulation and differentiation of memory B cells to PCs or whether they are derived from intrinsically long-lived PCs. Several studies are in favor of the first hypothesis that persistent antigen or infection and polyclonal memory B cell activation is required (18–22). Nevertheless, evidence is growing that PCs can persist in the absence of continuous stimulation (23–25). It was shown that PCs require cell-intrinsic and extrinsic survival signals such as cytokines and adhesion molecules from nursery cells like monocytes, eosinophils, and megakaryocytes for longterm survival in BM niches (26–29). Once they reach the BM and successfully compete for a niche, PCs have a lifespan varying from a few months to years and even decades during which they constantly secrete antibodies (30, 31). In contrast to PCs, which do not express surface Ig, MBCs respond to secondary Ag encounter. They exhibit the intrinsic ability to respond with a proliferative burst faster compared to naïve B cells (32) and were found to seed new GCs and/or differentiate into PCs (33–37). Antibody responses generated during secondary responses are usually of higher affinity for the cognate Ag compared to those of a primary response.

We have previously shown that immunization with VLPs derived from the RNA bacteriophage Qβ elicit strong and sustained IgG antibody responses by activation of MZ and follicular B cells with the latter forming GCs (38–40). MBCs and PCs were rapidly generated and detectable as early as 3 days and up to several months after immunization in spleen and BM (41, 42). Here we show, that MBCs generated against Qβ proliferated during Ag recall experiments but exclusively differentiated into secondary PCs and failed to respond to multiple rounds of Ag stimulation. Secondary PCs exhibited the unique ability to produce 30 times more antibodies of increased affinity compared to primary PCs. The secondary PCs were found in spleen as well as in BM early on day 4 but almost completely disappeared by day 6 after Ag re-encounter from both organs. In addition, antibodies produced by secondary PCs were cleared from the system within weeks indicating that secondary PCs are functionally short-lived. Inducing MBCs that differentiate into secondary PCs by vaccination could represent a novel pathway for efficient and rapid control of infectious diseases by the induction of an early wave of high affinity antibodies.

### MATERIALS AND METHODS

#### Study Design

The goal of this study was to further characterize secondary PCs, which were generated by MBCs after Ag challenge. To achieve this, adoptive transfers in allotypic mice (Ly5.1/Ly5.2 and IgHa/IgHb) were performed. This enabled us to study primary and secondary immune responses in the same animal. All mice were kept according to Cantonal Veterinary guidelines at the central animal facility (Department for BioMedical Research) of the University of Bern and controlled laboratory experiments were performed in accordance with ethical principles and guidelines of the Cantonal Veterinary Office Bern, Switzerland. Animals were randomly assigned to the different groups. MBCs were generated by VLP immunization of mice. The control naïve mice remained untreated. At the same time, B cells were isolated from memory and naive mice and transferred into recipients. Upon immunization with VLPs, serum samples, spleens, and BM were collected and subjected to ELISA, ELISPOT, and FCM analysis. The investigators who performed the experiments, assessed, analyzed, and quantified the results were not blinded and aware of which group a sample was taken from. Individual groups consisted of 4–5 mice. All experiments were performed in at least 2 independent biological replicates, apart from intracellular FCM analysis of PCs at day 6 after challenge. Data were collected at previously determined time points. All data were included in the analysis.

#### Mice

C57BL/6JRccHsd wildtype mice were purchased from Envigo (Horst, The Netherlands). The IgHa (B6.Cg-Gpi1 Thy1 Igh (Stock No. 001317)) mouse strain was purchased from the Jackson Laboratory (USA). We thank Prof. Annette Oxenius for the kind donation of the Ly5.1 (B6.SJL-Ptprc Pepc/BoyJ) mouse strain.

#### Antigen

The bacteriophage derived Qβ virus-like particles (VLPs) selfassemble and enclose bacterial RNA during their production in E. coli. Due to their particulate and repetitive structure, the VLPs are highly immunogenic. The purification process is described elsewhere (43).

#### Immunization

To induce primary immune responses and generate MBC against the VLPs, mice were immunized intravenously (i.v.) with 50 µg Qβ VLPs. To challenge adoptively transferred MBC or naive cells, recipient mice were immunized with 50 µg Qβ VLPs i.v. For intravenous administration the VLPs were formulated in 150 µl sterile PBS.

#### Adoptive Transfer

MBCs were generated by immunization of congenic donor mice (Ly5.1 or IgHa). At least 8 weeks after immunization donor mice were sacrificed and spleens isolated in RPMI media containing 2% FCS and antibiotics. A single cell suspension of the spleens was prepared and red blood cells were lysed using ACK buffer (0.15 M ammonium chloride, 0.01 M potassium hydrogen carbonate, pH 7.2–7.4). The splenocytes were PNA− and B220+ MACS purified. For PNA negative purification splenocytes were labeled using PNA-biotin (Vector Labs, B-1075) and PNA+ cells were depleted by Strepravidin MicroBeads (Milteny Biotec, 130-048-101) according to the manufacturer's protocol. Positive selection using B220 MicroBeads (Milteny Biotec, 130-049-501) was performed according to the manufacturer's protocol.

Purified cells from 1/3 of a donor spleen (Ly5.1 or IgHa; ∼1– 3 × 106 cells) were adoptively transferred i.v. into congenic host mice (Ly5.2 or IgHb). Control mice received PNA− and B220+ purified splenocytes from naïve congenic mice. One day after MBC transfer host mice were challenged with 50 µg Qβ VLPs i.v.

#### Flow Cytometry (FCM)

For FCM staining tissues (spleen, BM, kidney, lymph nodes (LN), liver, lung) of mice after adoptive transfer were isolated in RPMI supplemented with 2% FCS and antibiotics and single cell suspensions were prepared. Blood was collected in phosphate buffer containing heparin (1–2 units/ml). Red blood cells were lysed using ACK buffer prior to staining. Fc receptors were blocked using an anti-CD16/32 antibody. Qβ specific class switched (CS) B cells were identified as IgM, IgD, CD4, CD8, GR1, CD11b, CD11c negative (all antibodies labeled with phycoerythrin (PE)), and positive for B220 labeled with PE-Cy7 and Qβ VLP labeled with Alexa Flour 488. To discriminate Qβ specific PCs from Qβ specific activated and CS B cells, surface immunoglobulins (Ig) of specific cells were blocked using unlabeled Qβ VLPs. PCs were further stained with and characterized as IgM, IgD, CD4, CD8, GR1, CD11b, CD11c negative (all antibodies labeled with PE) and B220-PE-Cy7 low. To detect Qβ specific PCs by intracellular staining of specific Ig, splenocytes were permeabilized using FACS lysing solution (BD, 349202) containing 0.04% Tween20 and stained with Alexa Flour 488 labeled Qβ VLPs. The congenic marker Ly5.1 (antibody labeled with APC or PerCP-Cy5.5) identified all transfer derived B cells. Dead cells were stained by the addition of propidium iodide solution (PI, Sigma, 10µg/ml) directly before acquisition. For detection of dead cells after fixation and permeabilisation, the Fixable Viability Dye eFluor 520 (eBioscience, 65-0867-14) was used according to the manufacturer's instructions.

Qβ VLPs were labeled with the Alexa Flour 488 protein labeling kit (Thermo Fisher Scientific, A10235) or Alexa Flour 647 NHS Ester (Thermo Fisher Scientific, A20006) according to the manufacturer's instructions.

Data acquisition was performed on a FACS Canto (BD) and analyzed using FlowJo V10.1 (Flowjo, LLC, USA). All antibodies were purchased from BD Biosciences and Biolegend.

#### ELISPOT

Spleens from mice after adoptive transfer were isolated and a single cell suspension was prepared. To collect BM cells, tibia and femur were flushed with RPMI media containing 2% FCS and antibiotics. After red blood cell lysis with ACK buffer, cell numbers of splenocytes and BM cells were determined using the Cellometer mini (Nexcelom, USA). 5 × 105 cells were seeded per well on MAIPS Elispot plates (Millipore, MAIPS4510) previously coated with 10µg/ml Qβ VLPs overnight at 4◦C and blocked with 2% BSA in PBS for at least 2 h. After performing a 2 fold dilution series cells were incubated for 5 h at 37◦C and 5% CO2. Subsequently cells were washed off and bound specific antibodies produced by PCs were detected using a goat antimouse IgG antibody (EY laboratories, AT-2306-2) followed by a donkey anti-goat alkaline phosphatase secondary antibody (Jackson Immunoresearch, 705-055-147). Spots were visualized by the AP Conjugate Substrate Kit (BioRad, 1706432) and counted using an EliSpot Reader (AID, Germany). The spot size was quantified with the EliSpot 7.0 iSpot software of the EliSpot Reader as the average surface area of the spot.

# CFSE Proliferation

To analyse the proliferation of transferred cells, the donor cells were labeled with CFSE (Biolegend, Cat No. 423801) after MACS purification and before transfer into congenic hosts, according to the manufacturer's protocol. FCM staining was carried out similarly as described above. In this case, Qβ specific CS B cells were detected with VLPs labeled with Alexa Flour 647.

#### Splenocyte Cell Culture

Spleens from mice that had received memory or naïve B cells were isolated 5 and 6 days after VLP challenge. A single cell suspension of splenocytes was prepared. After red blood cell lysis with ACK buffer, cell numbers of splenocytes were determined using the Cellometer mini (Nexcelom, USA). 10 × 106 cells were seeded in 1 ml RPMI media containing 10% FCS and antibiotics per well in 24 well plates (Falcon Multiwell, Corning). The cells were incubated for 72 h at 37◦C and 5% CO2. Cell supernatants were harvested and the antibody content determined by ELISA.

### ELISA

Serum samples were obtained from blood collected at the indicated time points during experiments using Microtainer tubes (BD, 365967). Corning half area 96 well-plates were coated with 50 µl of 1µg/ml Qβ VLPs overnight at 4◦C. Sera were 1:10 pre-diluted and 1:4 further serial diluted to analyse a total of 7 dilutions per sample. Qβ specific antibodies were detected using mouse anti-mouse IgG for both allotypes. IgHaspecific (biotin ms anti-ms IgG1[a] (10.9), biotin ms anti-ms IgG2a[a] (8.3) from BD) and IgHb-specific (biotin ms anti-ms IgG1[b] (B68-2), biotin ms anti-ms IgG2a[b] (5.7) from BD) antibodies were detected using horseradish peroxidase (HRP) labeled streptavidin (Dako).

Cell supernatants were used undiluted and a 1:2 serial dilution was performed. An anti-Qβ monoclonal antibody (purified from hybridoma cells) was used as a standard to quantify specific antibodies in the supernatants. Qβ specific antibodies were detected using goat anti-mouse IgG-HRP (Jackson ImmunoResearch, 115-035-071).

The absorbance readings of the tetramethylbenzidine (TMB) color reaction at 450 nm for the serum samples were interpreted as OD50 antibody titers. The OD50 antibody titers are defined as the reciprocal of the dilution that reaches half of the OD max. The anti-Qβ monoclonal antibody standard curve was used to calculate antibody concentrations in the cell supernatants.

#### Avidity ELISA

Serum samples were obtained from blood collected at the indicated time points during experiments using Microtainer tubes (BD, 365967). Corning half area 96 well-plates were coated with 50 µl of 1µg/ml Qβ VLPs overnight at 4◦C. Sera of the different time points were applied with a 1:20 pre-dilution and 1:4 further serial diluted. After 1 h incubation, the sera were washed off and the plates washed 3 times 5 min either with 7M urea in PBST (PBS containing 0.05% tween 20) or PBST only. Qβ specific antibodies were detected using mouse antimouse IgG for both allotypes. IgHa-specific (biotin ms antims IgG1[a] (10.9), biotin ms anti-ms IgG2a[a] (8.3) from BD) and IgHb-specific (biotin ms anti-ms IgG1[b] (B68-2), biotin ms anti-ms IgG2a[b] (5.7) from BD) antibodies were detected using horseradish peroxidase (HRP) labeled streptavidin (Dako). The absorbance readings of the tetramethylbenzidine (TMB) color reaction at 450 nm served as basis for avidity index calculation. The avidity index (AI) was calculated by AIx = OD (dilution x) + urea / OD (dilution x)–urea.

# Antibodies/Reagents

#### Statistics

Statistical analysis was performed using GraphPad Prism Version 7.01 (GraphPad Software, USA). Statistically significant differences between two groups were calculated using unpaired t-tests. Statistically significant differences between more than 2 groups were determined using a one-way ANOVA followed by Tukey's or Sidak's multiple comparisons test. Statistical significance was defined as p < 0.05. The best fitting line was calculated by linear regression.

## RESULTS

#### Memory B Cell Derived Secondary PCs Produce Antibodies of Higher Avidity

We have previously shown that MBCs are generated against Qβ VLPs in a T cell-dependent manner (35, 38, 39, 44, 45). During secondary responses, these MBCs do neither extensively proliferate nor join GC reactions (35). T cell help, however, is essential for low-level MBC proliferation but dispensable for differentiation to secondary PCs during secondary immune responses (44). To reveal insights in the mechanism and kinetics of secondary PC formation from MBCs after antigenic restimulation, adoptive transfer experiments using congenic mice were performed (**Figure 1A**). To this end, MBCs were generated by immunizing donor mice (Ly5.1 or IgHa) with 50 µg Qβ VLPs. Eight weeks post immunization, splenocytes from donor mice were isolated and PNA− and B220+ MBCs were purified