Mechanisms underpinning poor antibody responses to vaccines in ageing.

Vaccines are a highly effective intervention for conferring protection against infections and reducing the associated morbidity and mortality in vaccinated individuals. However, ageing is often associated with a functional decline in the immune system that results in poor antibody production in older individuals after vaccination. A key contributing factor of this age-related decline in vaccine efficacy is the reduced size and function of the germinal centre (GC) response. GCs are specialised microstructures where B cells undergo affinity maturation and diversification of their antibody genes, before differentiating into long-lived antibody-secreting plasma cells and memory B cells. The GC response requires the coordinated interaction of many different cell types, including B cells, T follicular helper (Tfh) cells, T follicular regulatory (Tfr) cells and stromal cell subsets like follicular dendritic cells (FDCs). This review discusses how ageing affects different components of the GC reaction that contribute to its limited output and ultimately impaired antibody responses in older individuals after vaccination. An understanding of the mechanisms underpinning the age-related decline in the GC response is crucial in informing strategies to improve vaccine efficacy and extend the healthy lifespan amongst older people.

Introduction

The human lifespan has increased dramatically over the past century, largely owing to improvements in healthcare, hygiene and reduced child mortality rates [1]. Unfortunately, this increase in lifespan does not always coincide with an increase in healthspan, the period of life free from illness and disability [2]. This is because ageing is often accompanied by a loss in the physiological function of different tissues, organs and systems, including the immune system. The age-related changes in the immune system result in defects in immune system function, which leads to increased susceptibility to infections in older individuals who are more likely to have poor health outcomes [3]. This was clearly demonstrated in the COVID-19 pandemic, where older individuals are more at risk of severe disease and death after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [4,5].

The primary purpose of vaccination is to protect people from (re)infections and/or to reduce the severity of disease, by limiting pathogen replication and spread in the body. As such, vaccination represents an important intervention to limit the burden of infectious diseases amongst older individuals and improve their health span [6]. However, there is clear evidence that ageing is associated with a decline in vaccine responses [7]. Both humoral and cell-mediated immunity are consistently poorer in older individuals compared to younger individuals post-immunisation [8], [9], [10], [11]. Furthermore, the longevity of the antibody response induced by vaccination also declines with age [12,13]. More recently, studies evaluating the immunogenicity of vaccine candidates against SARS-CoV-2 in older individuals also show that antibody responses are generally lower in older people after one dose of vaccine, though this can be boosted to levels comparable to those in younger people with a second dose [14], [15], [16]. An understanding of the mechanisms underpinning the age-related decline in vaccine responses is therefore crucial in informing new strategies to improve vaccine efficacy and to support healthy ageing.

How vaccine-induced humoral immunity is generated and how it changes with age

Vaccines represent one of the most impactful medical interventions in human history. By mimicking natural infection, most vaccines work by promoting the generation of pathogen-specific, long-lasting antibodies [17]. These antibodies confer protection against infections by binding to the pathogen to block it from establishing an infection, and recruiting other immune cells to promote its destruction. In addition, vaccines also induce the formation of pathogen-specific memory B and T cells, which serve as a second line of defence when the invading pathogen circumvents the antibody response [18]. These memory cells help to control an active infection by rapidly proliferating and differentiating into effector cells. Specifically, memory B cells rapidly differentiate into antibody-secreting plasma cells and can re-enter the germinal centre (GC) for further expansion and diversification [19]. Memory CD8+ T cells differentiate into effector CD8+ T cells that directly attack and kill virus-infected cells, while memory CD4+ T cells support multiple different immune cell types and help promote effective CD8+ T cell and antibody responses [20]. Such cell-mediated immunity has been shown to complement antibody-mediated protection in ensuring robust vaccine efficacy [21,22]. While both humoral and cellular immunity are important for ensuring vaccine efficacy, the production of protective antibody responses is typically considered the main correlate of vaccine-induced immunity [23], [24], [25]. This review will focus on how ageing influences the formation and maintenance of antibody-mediated immunity upon vaccination.

Vaccine-induced antibodies can be generated from two main cellular pathways: the extrafollicular response and the GC reaction. The extrafollicular response forms early post-immunisation, where antigen-primed B cells differentiate into plasmablasts in the extrafollicular foci of the spleen or in the medullary cords of lymph nodes [26]. These extrafollicular plasmablasts undergo expansion and differentiation to form short-lived plasma cells which secrete protective antibodies to control infection [27]. Since these plasma cells are short-lived, the extrafollicular response, whilst important for controlling the early stages of active infections, does not confer long-term immunity [28].

The main source of long-lived antibody-secreting plasma cells is the GC reaction. GCs are specialised microanatomical structures that appear later in the immune response in the follicular regions of secondary lymphoid organs [29]. The mature GC is made up of two distinct zones: the dark zone and the light zone. In the dark zone, GC B cells, known as centroblasts, undergo somatic hypermutation (SHM) of the antigen-binding variable regions of their immunoglobulin genes, which involves the action of the activation-induced cytidine deaminase (AID) enzyme [30,31]. Due to the random nature of the SHM process, the GC B cells then undergo a process of selection in the light zone to ensure that B cells carrying functional, high-affinity B cell receptors (BCRs) are preferentially selected while B cells with potentially autoreactive BCRs are eliminated. Light zone B cells, known as centrocytes, test their mutated BCR by binding and internalising antigens held on the surface of follicular dendritic cells (FDCs), before presenting processed antigens to T follicular helper (Tfh) cells on major histocompatibility complex (MHC) class II molecules [32]. The selection process is driven by cognate interactions with limited numbers of Tfh cells within the GCs [33,34]. GC B cells that are able to engage in cognate interactions with Tfh cells receive positive signals from Tfh cells via CD40/CD40L ligation and cytokines like IL-21 and IL-4, allowing them to preferentially survive within the GC [35], [36], [37]. These positively selected GC B cells upregulate c-Myc, enabling return to the dark zone and further rounds of proliferation and SHM [38]. GC B cells carrying lower-affinity or autoreactive BCRs fail to engage Tfh cells in cognate interactions to receive survival signals, causing them to undergo apoptosis. Their corpses are then cleared by tingible body macrophages [39,40]. Positively selected GC B cells eventually differentiate and exit the GC as long-lived antibody-secreting plasma cells or memory B cells.

After leaving the GC, most long-lived plasma cells migrate to the bone marrow via mechanisms involving the CXCR4/CXCL12 chemokine axis and sphingosine-1-phosphate receptor 1 expression [41,42]. These plasma cells reside in survival niches in the bone marrow, where they continue to produce high titres of antibodies [43], [44], [45]. Conversely, memory B cells circulate in the blood, secondary lymphoid tissues and bone marrow and are able to differentiate into plasma cells with greater efficiency than naïve cells upon secondary exposure to antigens, contributing to anamnestic antibody responses [46]. As such, the GC is important in generating long-lasting humoral immunity and is crucial in ensuring vaccine efficacy.

Many other cell types are involved in the initiation and maintenance of the GC response; these include stromal cell subsets such as follicular reticular cells (FRCs) that regulate the recruitment, localisation and survival of T and B cells early in the immune response, and the network of FDCs and CXCL12-producing reticular cells (CRCs) within the B cell follicle, which are crucial for maintaining the structural integrity of the GC (reviewed in [47,48]). The GC is also regulated by T follicular regulatory (Tfr) cells, which most studies indicate have immunosuppressive functions [49], [50], [51], [52], [53], [54]. As such, the GC reaction requires the complex interplay of many different cell types, regulated in a time- and space-sensitive manner, in order to generate protective humoral immunity. Given the collaborative nature of the response, alterations in a single cell type, or in multiple GC players may be responsible for the diminution of the size and quality of the GC response in ageing.

Since the GC plays a vital role in generating vaccine-specific antibodies, it is important to understand how ageing influences the GC reaction, to allow us to decipher the mechanisms underlying the age-related decline in vaccine responses. Ageing has been associated with a decline in the number and size of GCs in human mesenteric and cervical lymph nodes, which are continually exposed to antigens [55]. In the context of vaccination, multiple studies have shown that the number, magnitude and volume of GC response is significantly lower in aged mice compared to young mice after immunisation [56], [57], [58], [59], [60]. The age-related decline in GC response is accompanied by a decline in the number of long-lived bone marrow-resident plasma cells and vaccine-induced antibody titres in aged mice, especially those that are high affinity and class-switched [58,59,61,62]. However, the formation of extrafollicular plasma cells upon immunisation does not seem to be affected by age [59].

Age-related defects in the GC response of older humans after immunisation are less well-characterised due to the limitations of accessing the draining secondary lymphoid tissues after vaccination . Circulating Tfh (cTfh) cells, which are cells in the blood and lymph that resemble GC Tfh cells and that are found in higher frequencies post-vaccination, have been identified as strong correlates of vaccine-induced antibody production in humans [63], [64], [65], [66], [67], [68], [69]. While their exact relationship to GC Tfh cells remains unclear, cTfh cells share transcriptional, epigenetic, and phenotypic characteristics with GC Tfh cells, and are clonally and functionally related to GC Tfh cells [64,[66], [67], [68], [69], [70], [71]]. In people aged 65 years old and older, the frequencies of hemagglutinin-specific cTfh cells in the peripheral blood are reduced post-influenza vaccination, which correlates with reduced hemagglutinin-specific antibody responses [58,72,73]. Nevertheless, the effect of age on the frequencies of lymph node GC Tfh cells upon immunisation in humans remains to be determined. The decline in GC response in older humans can also be deduced by a reduction in its outputs, which include vaccine-specific antibody production and antigen-specific memory B cell formation. In particular, the vaccine response in older people is characterised by lower titres of vaccine-specific antibodies, an increase in self-reactive antibodies, and a shorter duration of the antibody response [12,13,74]. Nevertheless, despite the quantitative decrease in binding antibody titres, most evidence suggest that the quality of vaccine-induced antibody response, in terms of antibody avidity or neutralisation capacity, is intact with age [75], [76], [77], [78]. This suggests that reduced antibody titre, rather than the functional capacity of the antibodies, is the primary change in vaccine-induced antibody response during ageing.

Memory B cell formation, which is another important output of the GC reaction, has also been shown to be impaired in older individuals. A recent study from our lab showed that there is impaired expansion of antigen-specific memory B cells in the peripheral blood of older adults post-influenza immunisation [79]. This age-related impairment in antigen-specific memory B cell formation was also observed after SARS-CoV-2 vaccination with mRNA-based vaccines [80]. As memory B cells are key for mounting robust recall responses by differentiating into antibody-secreting plasma cells with greater efficiency or by initiating secondary GCs [19,81], poor production of antigen-specific memory B cells will likely contribute to poor recall responses in older individuals. Overall, the evidence points to defects in the GC response in older individuals, which results in impaired antibody production and memory B cell formation during vaccination (Fig. 1).

Fig. 1

A summary of some known mechanisms underpinning defects in the germinal centre (GC) response during ageing, which eventually contribute to poor vaccine responses. (DC, Dendritic cell; FDCs, Follicular dendritic cells; FRCs, Follicular reticular cells; Tfh, T follicular helper ; Tfr, T follicular regulatory) During immunisation with a T-dependent antigen, naïve antigen-specific CD4+ T cells are first primed by DCs that present processed antigen:MHCII and provide signals to stimulate Tfh differentiation. However, in aged mice, migratory conventional type 2 dendritic cells (cDC2s) have impaired activation due to reduced type I interferon (IFN-I) signalling [58]. This in turn results in defects in T cell priming and impairments in the Tfh maturation process [58,135]. The reduced production of chemokines by stromal cells like FRCs have been proposed to contribute to the defective migration of T cells and antigen-bearing DCs to the T cell zone, further contributing to poor T cell priming [107,135]. While alterations in TCR signalling in aged naïve T cells results in an accumulation of pre-Tfh cells, there is a block in the differentiation of pre-Tfh cells into mature GC Tfh cells during ageing, resulting in fewer antigen-specific Tfh cells in the GCs of aged mice [60,136]. This contributes to a higher Tfr:Tfh ratio observed in the GCs in aged mice and poorer T cell help provided for GC B cells [132]. B cells from aged mice and humans do not have intrinsic defects in responding to stimulation and differentiating into plasma cells [61, 219, 220]. However, age-related defects in class-switch recombination of activated B cells have been shown [59,188,222]. While the mechanism of somatic hypermutation appears to be unperturbed with age [226], B cells from aged humans have been shown to acquire fewer de novo somatic mutations post-immunisation [209,227], which might be the result of a smaller GC reaction and/or the preferential recruitment of memory cells that requires fewer mutations. In aged mice, FDCs have intrinsic defects in activation and expansion, and are impaired in immune complex retention, which contributes to a lower magnitude of the GC response [151,166]. Defects in FDC function and Tfh cell help can contribute to impairments in the positive selection process of GC B cells [79,209,226]. Eventually, an impaired GC response results in poor memory B cell [79,228] and long-lived plasma cells formation and reduced vaccine-induced antibody titres in older individuals [7,12,58,59,74,77].

Mechanisms underpinning the age-related decline of the GC response

Since GCs require the function and interactions of different cell types, the age-related defects in the GC reaction could plausibly be caused by changes, with varying levels of contribution, in multiple cell types. In this section, we will discuss how ageing influences the phenotype and function of T and B cells and the GC microenvironment, that can contribute to the age-related impairment in the GC response to vaccination.

Changes in the CD4+ T cell compartment and function with age

Age-related defects in the CD4+ T cell response have been implicated as key factors contributing to the impaired GC response in aged individuals. The transfer of aged T cell receptor (TCR)-transgenic T cells into young CD4-deficient hosts results in a lower magnitude of the GC response and reduced antigen-specific antibody production after immunisation, compared to the transfer of T cells from a young donor mouse [82]. Correspondingly, the attenuated GC response in aged mice can be rescued to levels similar to that of young mice, by supplementing them with young polyclonal or TCR-transgenic CD4+ T cells [60,82]. In addition to the reduced size of the GC reaction, aged CD4+ T cells have been implicated in reduced SHM and impaired selection of GC B cells, which are essential for affinity maturation [62]. These observations suggest that ageing results in the reduced ability of CD4+ T cells to contribute efficiently to the GC response. These defects may be attributed to impairments in aged T cells in differentiating into Tfh cells and/or defects in their helper function.

Age-related changes to the composition of the CD4+ T cell compartment

Tfh cells form from naïve CD4+ T cells upon first exposure to a particular antigen. As such, age-related changes in the naïve T cell compartment have the potential to affect Tfh cell differentiation and function during ageing. Age-associated thymic involution, characterized by structural alterations and functional decline of the thymus, results in a decrease in the output of naïve cells with new TCRs [83]. This has a larger effect on naïve T cell numbers in mice, in which maintenance of the peripheral naïve T cell compartment is believed to be dependent on thymic output [84]. In contrast, the naïve T cell pool in adult humans is maintained by homeostatic peripheral T cell division [84]. Homeostatic proliferation has been shown to be efficient in maintaining a sufficiently diverse and functional naïve CD4+ T cell pool with age, based on studies that performed deep sequencing of the TCRβ chains in naïve CD4+ T cells from the peripheral blood and secondary lymphoid tissues of human donors [85], [86], [87]. Nevertheless, an age-related contraction of the naïve T cell pool is still observed in older mice and humans due to continuous antigen exposure that promotes a shift in the T cell subset distribution from naïve T cells to memory subsets [88,89]. A recent single-cell transcriptomic and proteomic analysis of splenic CD4+ T cells showed a contraction of the naïve T cell compartment and the accumulation of distinct activated regulatory, exhausted, and cytotoxic CD4+ T cell subsets in aged mice [90]. Since this study involved unimmunized mice, the characterized age-related remodelling of the CD4+ T cell compartment is not due to an acute immune response, but likely driven by factors such as the chronic low-grade systemic inflammation that develops with age, known as inflammaging. Indeed, the age-associated T cell subsets identified had higher gene expression of inflammatory molecules and their frequencies correlated with increased concentration of pro-inflammatory cytokines like IL-6 and IL-27 [90]. The accumulation of terminally differentiated and exhausted CD4± Tcells with age have been implicated in immune function decline, due to their defects in proliferation and loss of effector functions like cytokine production [91]. Recently, Sayed et al. constructed a metric for age-related inflammation (iAge) by applying deep learning methods on blood immune biomarkers that was able to predict multiple age-related morbidities, including decline in immune function [92]. Together, the evidence suggests that inflammaging drives the accumulation of terminally differentiated T cell subsets that results in poor T cell function and that can potentially contribute to impaired vaccine responses in older people, although the actual causal links remain to be characterized. There is also some evidence that naïve cells with high affinity to self-antigens are preferentially retained, potentially leading to increased autoimmune susceptibility with age [93,94].

Age-related changes to CD4+ T cells phenotype and function

Apart from changes to the proportions of T cells subsets, ageing also results in changes in T cell phenotype and function, which have been associated with higher risk of autoreactivity and poorer ability to respond to foreign antigens efficiently during infection or immunisation. Naïve CD4+ T cells from aged mice have an accumulation of damaged DNA, which in turn potentiates TCR-induced proliferation and promotes age-related autoimmune inflammation in vivo [95]. In the context of stimulation with foreign antigens, naïve CD4+ T cells from aged mice and humans display reduced proliferative capacity and IL-2 production, and generate effector cells with poorer cognate helper function [96], [97], [98]. These seemingly contradicting results for the effects of age on T cell proliferation suggest that there are possibly defects that prevent aged T cells from engaging efficiently with antigen and/or changes in signalling pathways that affect their proliferation and differentiation into effector cells during infection or immunisation.

Multiple molecular mechanisms that contribute to these functional defects in T cells from aged individuals have been characterised. Activation defects of T cells from aged mice have been attributed to the hyperglycosylation of surface glycoproteins and altered cytoskeletal assembly that prevent them from forming functional synapses with antigen-presenting cells [99]. In humans, naïve CD4+ T cells from older individuals have reduced expression of the transcription enhancer YY1 and consequently reduced miR-181a expression, that results in lower ERK activity and defects in TCR sensitivity and signal strength [100,101]. Impaired TCR activation may in turn alter T cell differentiation and function upon stimulation [100,102]. Naïve T cells from older people have also been reported to have increased miR-21 expression, which results in the sustained activation of multiple pathways, such as ERK, AP-1, and AKT-mTORC signalling upon T cell activation, and this consequently modulates the transcriptional networks governing T cell differentiation to favour differentiation towards inflammatory effector T cells over protective memory cell phenotypes [103]. In turn, sustained mTORC1 activation of naïve CD4 T cells from older people in late endosomes has been associated with proliferative defects and is proposed to contribute to the impaired differentiation of aged naïve T cells into Tfh and protective memory cell subsets during infection [104]. It has also been reported that naïve transgenic T cells from aged mice are defective in upregulating CD40L expression after antigen stimulation in vitro, compared to those from young mice [82]. Similarly, CD4+ memory T cells from older people have been shown to be defective in upregulating CD40L, CD69 and ICOS and in IL-4 and IL-21 production after in vitro CD3/CD28 stimulation, due to increased and sustained expression of dual-specific phosphatase 4 [105]. Since these receptors and cytokines are important for Tfh cell differentiation and function, their reduced expression suggest that ageing may result in cell-intrinsic defects in T cell activation, which can affect Tfh cell differentiation and help provision for B cells [105,106]. Collectively, the data suggests that intrinsic changes in naïve and memory T cells phenotype with age can alter its activation and differentiation trajectory, which can have a negative impact on vaccine response in older people.

Naïve CD4+ T cells from aged mice also show cell-intrinsic defects in migrating into draining lymph nodes at the diapedesis step, when transferred into young adult mice, and display lower motility in the parenchyma compared to those from young mice, which can collectively contribute to reduced numbers of activated T cells in the lymph nodes [107]. Together, CD4+ T cells from older individuals have intrinsic defects in TCR-induced activation and defects in migration, which may negatively affect Tfh cell differentiation and contribute to a reduced or delayed GC response during immunisation in ageing.

Age-related defects in Tfh differentiation and function

The development of mature Tfh cells involves a stepwise process. The first step involves priming of naïve CD4+ T cells via peptide:MHCII presentation by dendritic cells (DCs) [108]. Multiple signals received by the primed CD4+ T cells help commit them to the Tfh lineage [109]. These signals include the interaction of inducible T-cell costimulator (ICOS) on T cells with ICOS-ligand on DCs [110], together with cytokines IL-12 and TGF-β (in humans) [111], [112], [113] and IL-6 and IL-21 (in mice), which activate STAT-3 [114,115]. These will induce the expression of transcription factors Bcl6 and Ascl2 in the Tfh cell precursors (pre-Tfh), which drives the upregulation of CXCR5 and the concomitant downregulation of CCR7 [116], [117], [118], [119]. This then promotes the migration of the pre-Tfh cells towards the B cell follicles which are rich in CXCL13, where the second step of Tfh cell development occurs [120]. Here at the T-B border, pre-Tfh cells engage in symbiotic interactions with B cells, which are essential for the differentiation of both cell types and the initiation of the GC response. B cells provide additional antigenic stimulation and costimulatory signals including CD40, ICOSL, CD80 and CD86, which help ensure the sustained expression of Bcl6 in the Tfh cells and promote further Tfh cell maturation [121], [122], [123]. In return, Tfh cells provide B cells with cytokines such as IL-21, and CD40L signals required for B cell proliferation and differentiation toward GC and extrafollicular fates [124,125]. Signals provided by Tfh such as IL-4, IL-10 and IFN-y are also important in supporting class-switch recombination of B cells at this stage [106,[126], [127], [128], [129]]. Tfh cells then move from the T-B border into the GC, becoming GC Tfh cells. In the GC, Tfh cells play an important role in the selection of GC B cells, by providing help to GC B cells that are able to engage them in cognate interactions [130]. The signals provided by GC Tfh cells, including CD40L, IL-21 and IL-4, will promote the survival, proliferation, and terminal differentiation of selected GC B cells into plasma cells and memory B cells [124,131].

Studies evaluating the impact of ageing on Tfh differentiation have yielded conflicting results. While some studies report an increase in Tfh differentiation in aged mice post-vaccination [132,133] and in older people [134], other studies have shown a decrease in Tfh differentiation in older mice [58,135] and in older people post-vaccination [58,72,73]. Some factors that might account for this discrepancy include different markers used to identify the Tfh cells, different flow cytometric gating strategies, whether Tfr cells are excluded from the Tfh cell population, and whether the studies were looking at antigen-specific Tfh cells or total Tfh cells. Multiple studies have observed that while ageing does not result in defects in the formation of pre-Tfh cells, there is an age-related impairment in their development into mature GC Tfh cells in mice [60,136]. A closer look into the underlying mechanism showed that antigen-specific CD4± Tcells in aged mice post-vaccination have a more regulatory phenotype, characterized by lower IL-2 and increased IL-10 production, and also have lower expression of Tfh cell markers like ICOS and CXCR5, indicating an age-related dysregulation in effector CD4± Tcell differentiation that might contribute to impaired mature Tfh cell formation [60,137].This is also observed for humans, where naïve CD4± Tcells from older donors were observed to have enhanced differentiation into pre-Tfh cells when stimulated via CD3/CD28 without Tfh-polarising cytokines IL-12 and TGFβ, compared to those from younger donors, but older people have impaired antigen-specific cTfh formation post-vaccination [73,136]. Further investigation into the molecular programs governing pre-Tfh cell formation revealed increased expression of the transcription factor RBPJ as an important factor in driving Notch-mediated early Tfh cell differentiation in aged naïve CD4± Tcells [136]. This suggests that how Tfh cells are defined may also contribute to the discordance between studies and that the observations showing no defects in pre-Tfh differentiation but impairments in the formation of mature GC Tfh cells might help resolve the seemingly contradicting data on the effects of age on Tfh cell formation.

In addition, ageing can also result in impairments in the formation of antigen-specific Tfh cells. Co-culture of Tfh cells from immunised mice with CD19+ B cells from a young donor in a non-antigen-specific assay showed that Tfh cells from aged mice were equally able to induce class-switch recombination and activation of B cells as those from young mice [132]. However, when the cocultures were performed in an antigen-specific assay, Tfh cells from aged mice showed cell-intrinsic defects in providing B cell help [132]. This was also shown in vivo, where transfer of Tfh cells from aged mice into young Cd28−/− mice resulted in reduced GC B cells and plasma cell differentiation as compared to transfer with cells from young mice, although the readout of in vivo experiments will also be susceptible to differences in Tfh cell migration [132]. The discrepancy between findings in antigen-specific and non-specific helper assays could be explained by the significantly lower frequencies of antigen-specific Tfh cells that form within the total Tfh cell pool in ageing, therefore providing a limited number of antigen-specific helper cells in these in vitro assays [58,60,132]. In humans, fewer antigen-specific cTfh cells have also been observed after influenza vaccination in older donors [73]. This suggests that ageing results in defects in the formation of antigen-specific Tfh cells during immunisation, which could be caused by a combination of T cell-intrinsic changes such as changes in TCR signalling, as previously discussed, and T cell-extrinsic factors such as a suboptimal priming environment or inflammaging [58,73,136,138].

CXCR5+ PD1+ pre-Tfh cells derived from older people have similar capacity as those from younger individuals in stimulating antibody production when co-cultured with allogeneic B cells from younger adult donors [136]. However, age-related defects have been observed in the ability of cTfh cells from older people in inducing IgG production by B cells in co-culture assays involving the Staphylococcal enterotoxin B superantigen, which induces non-specific polyclonal T cell activation [72]. This may suggest specific changes in TCR sensing in aged cTfh cells, that prevents efficient interaction with superantigens. Together, the evidence thus far suggests that ageing results in a reduction in the development of antigen-specific Tfh cells and cell-intrinsic changes in TCR sensitivity, which may limit their ability to provide B cell help and contribute to poor humoral response during vaccination.

After seasonal influenza vaccination, cTfh cells from older people have gene expression signatures enriched for pro-inflammatory pathways such as TNFα signalling and IL-2 signalling [73,139]. This may suggest that ageing alters the way Tfh cells sense their environment, or that inflammaging may negatively impact Tfh cell formation or function in a cell-extrinsic way. It has also been reported that ageing is associated with an accumulation of IL-10-producing Tfh cells in aged mice and humans, which contributes to increased systemic levels of IL-10 and is proposed to be a mechanism to control IL-6-driven inflammaging [137]. This age-related increase in IL-10 levels contributes to limited vaccine responses in aged mice as blocking the IL-10 receptor was able to reverse age-related defects in vaccine-specific antibody production and B cells numbers [137]. This suggests that the dampened T cell responses to infection or immunisation can be an adaptive mechanism to cope with inflammaging, at the expense of a higher vaccine response [140]. Correspondingly, the addition of pro-inflammatory cytokines (TNFα, IL-1, and IL-6) in vaccine preparation has been shown to enhance the cognate helper function of both young and aged donor CD4+ T cells in young recipient mice, which translated to improved vaccine-specific antibody production [141]. Collectively, this suggests that extrinsic factors in the aged environment such as inflammaging can affect the cytokine profile and helper function of Tfh cells and the addition of pro-inflammatory stimuli may be beneficial in boosting vaccine responses in older individuals. `

Age-related changes in Tfr cells

Another specialised subset of CD4+ T cells involved in the GC response is Tfr cells. Tfr cells are derived from thymic Foxp3+ regulatory T (Treg) cells [49], [50], [51] or from Foxp3- naïve T cells in the periphery, via the induction of Foxp3 expression prior to Tfr differentiation [142]. Tfr cells express Tfh-associated molecules, including CXCR5, ICOS, PD-1 and Bcl6 [50,143]. They however do not express helper cell factors like CD40L, IL-21 and IL-4, and share features of Treg cells, such as the expression of CTLA-4, Foxp3 and GITR [49], [50], [51]. Tfr cells function to regulate class switch recombination, the GC response and autoimmunity [[49], [50], [51], [52], [53], [54],144] .

Ageing has been associated with an accumulation of Treg cells in both humans and mice, which has been hypothesised to limit the immune response in ageing [90,[145], [146], [147], [148]]. This contributes, in part, to a preferential accumulation of Tfr cells over Tfh cells in aged mice post-immunisation, resulting in a higher Tfr:Tfh ratio [132]. It is unlikely, however, that an age-related increase in Tfr:Tfh ratio can affect the GC as reducing Tfr cell numbers by half, using a Cxcr5fl/fl;Foxp3cre mouse model, has been shown to have no impact on the size or output of the GC [149]. Moreover, 18-month-old Cxcr5fl/fl;Foxp3cre mice do not have an improved GC response upon vaccination compared to their aged littermates, despite having fewer Tfr cells, indicating that Tfr numbers alone do not cause a diminished GC response in ageing (MAL, unpublished). Tfr cells from young and aged mice have also been shown to have equal immunosuppressive function in in vitro assays, though Tfr cells in aged mice have some phenotypic differences, including higher PD-1 expression and reduced ICOS expression [132]. Nevertheless, how the age-related accumulation of Tfr cells and their phenotypic changes contribute to poor vaccine responses in ageing remains to be fully understood.

Ultimately, ageing results in changes in the proportions of CD4+ T cell subsets and intrinsic changes in T cells, which in turn affects their differentiation into Tfh cells and their effector function (Fig. 1). Defects in T cell function contribute to a decline in the quantity and quality of the GC response and consequently poorer vaccine responses. As such, strategies to boost T cell function by targeting specific signalling defects will be beneficial in boosting vaccine responses in older individuals. Apart from cell-intrinsic defects in T cell development and function, much evidence has also pointed to the role of age-related defects in the microenvironment on the GC response.

Age-related defects in the stromal cell network

Studies involving the adoptive transfer of young CD4+ T cells into young adult or aged host mice have consistently shown that an aged microenvironment contributes to impaired migration, expansion and differentiation of transferred T cells, which results in poorer GC responses post-immunisation [58,135,150]. Consistent with these transfer studies, heterochronic parabiosis experiments support a role for the aged microenvironment in contributing to the poor initiation of the GC reaction [151]. One of the key components of the microenvironment is the stromal cells that provide the scaffold upon which immune cells enact their response to immunisation. There are multiple types of stromal cells in secondary lymphoid tissues. FRCs in the T cell zone secrete CCL19 and CCL21, which are important in the homing of naïve T cells and antigen-bearing DCs to the T cell follicles, via CCR7-dependent recruitment [47,152,153]. FRCs are also important in maintaining lymph node homoeostasis and are indispensable in the initiation of a GC response [154]. The key stromal cell type involved in the structure and function of the mature GC are the FDCs. In the absence of an immune stimulant, FDCs are located in the primary B cell follicle (one that does not contain a GC) and play an important role in antigen capture and display, which facilitates the activation of B cells [155]. FDCs are also a major source of the B cell chemoattractant CXCL13, which promotes the homing of B cells to the follicle [156]. During initiation of the GC response, CXCL13 provision by FDCs and CCL19/CCL21 provision by FRCs help ensure the localization of CXCR5+ CCR7+ T and B cells to the T:B border and interfollicular area to promote cognate interactions [47]. After immune challenge, FDCs reside in the light zone of the mature GC in the secondary follicle, where they, together with the dark zone-localised CRCs, regulate the transit of GC B cells between the dark and light zones to facilitate iterative rounds of B cell SHM and selection in the GC [157]. FDCs in the light zone capture and display immune complexes, which is crucial for the maintenance of the GC response and in promoting affinity maturation, by delivering antigens to GC B cells [158], [159], [160].

The aged microenvironment is characterized by an altered structural organization and reduced production of homoeostatic chemokines by stromal cells, which has been correlated with an impaired recruitment of T and B cells to secondary lymphoid organs [135,161,162]. Specifically, the lymph nodes of aged mice have reduced numbers of FRCs and FDCs [163], [164], [165]. This likely contributes to the lower expression levels of CCL19 and CCL21 in the draining lymph nodes and spleens of aged mice during immunisation, which is in turn correlated with an impaired recruitment of young donor antigen-specific CD4+ T cells and lower recovery of young cells in aged recipient mice during adoptive transfer experiments [107,135]. In addition, secondary lymphoid tissues of aged mice have lower CXCL13 expression than those of younger adult animals due to a reduction in the FDC network [107,161,164]. These changes have been proposed to contribute to the defective homing of T and B cells to the T:B border during vaccination in aged mice [107,161]. The interactions between T and B cells at the T:B border are essential for the maturation of both cell types to initiate the GC reaction [120]. As such, impaired localisation of T and B cells in secondary lymphoid tissues during immunisation, due to defects in the stromal-derived chemokine gradients, will have an impact on the development and function of the GC response, thereby contributing to the age-related defects in immune responses. In addition to reduced chemokine production, the altered structural organization of the FDC and FRC networks in aged mice also potentially contribute to their impaired ability to support naïve T cell survival, by limiting their access to antigens and survival factors like IL-7 [163]. Thus, this can result in the accelerated depletion of the naïve T cells pool in aged secondary lymphoid tissues and reduced activation of naïve T cells, contributing to defective immune responses in aged individuals.

Aged stromal cells are also impaired in responding to immune challenge. Aged FRCs and FDCs have been shown to have reduced proliferation and delayed expansion post-activation [151, 162]. Ageing also results in impairment of FDCs in capturing and retaining antigen, due to lower expression of FcγRIIb and costimulatory molecules like FDC-M2 [56,164,166,167]. This in turn limits their ability to support GC formation and promote antibody production, contributing to an age-related defect in the GC response [151, 168]. Collectively, the age-related reduction in stromal cell numbers, dysregulated chemokine production by the aged stromal network, their reduced response to immunisation and impaired intrinsic function such as defective antigen presentation likely contribute to defective homing of the relevant cells and poor GC responses in aged mice (Fig. 1).

Age-related defects in dendritic cells priming

Another key factor in the aged microenvironment that can contribute to age-related defects in T cell responses post-immunisation is defects in the priming process by DCs. Multiple subsets of DCs, including migratory or tissue-resident type 1 conventional dendritic cells (cDC1s), cDC2s and Langerhans cells, have been implicated in the priming of T cells [169], [170], [171], [172], [173]. In particular, migratory cDC2s have been proposed to be the dominant inducers of Tfh cell polarisation during vaccination [58,108,174]. Early after immunisation, cDC2s that bear the vaccine antigen are present in significantly lower numbers in lymph nodes of aged mice, compared to younger mice [58]. In addition, antigen-bearing cDC2s from aged mice had reduced presentation of peptide:MHCII complexes and expression of costimulatory ligands [58]. This defective response of cDC2s to vaccination in aged mice is a result of reduced production of type I interferon (IFN-I), likely derived from plasmacytoid DCs and macrophages. Boosting the production of IFN-I at the time of immunisation was able to correct the age-dependent defects in cDC2 priming and increase Tfh cell numbers in aged mice [58]. This suggests that cDC2s in aged mice have defects in activation and in their ability to provide costimulatory signals, which are essential to prime T cells, thereby contributing to impaired Tfh formation in aged mice. The important role for DCs in the defective immune response to vaccination is corroborated by studies that showed that transfer of young DCs into aged mice could restore, to some extent, the defects in CD4+ T cell activation in an aged microenvironment and boost the GC response after vaccination [135,150]. Furthermore, the transfer of TLR-activated DCs into aged mice has been shown to restore the ability of aged naïve CD4+ cells to differentiate into Tfh cells and consequently increase GC B cells generation and antibody production via IL-6 production [175]. As such, vaccine strategies that aim to co-activate DCs, such as via the delivery of antigens linked to antibodies or aptamers specific for DC receptors such as DEC-205 or the inclusion of TLR agonists, can be explored to improve vaccine antibody production in aged individuals [176,177]. Together, the data suggests that an aged microenvironment (including the stromal network and dendritic cells) can contribute to defective T cell differentiation and function in older individuals, resulting in poor GC response (Fig. 1). Importantly, some of these defects can be reversed or targeted to boost antibody responses in older individuals [58,175].

Changes in the B cell compartment and function with age

Studies investigating antibody responses after SARS-CoV-2 and seasonal influenza virus vaccination consistently show impaired antibody responses in older people and aged mice after primary immunisation [16,59,178]. Since these vaccines are likely to activate naïve B cells, it is important to ask if there are any changes in the B cell compartment and function that can contribute to impaired vaccine responses in older individuals. The number and composition of B cell populations are altered with age. Total CD19+ B cells numbers in human peripheral blood and bone marrow decline during ageing [179,180]. This is contributed in part by reduced lymphocyte production by the bone marrow, due to the increased myeloid-bias of aged bone marrow hematopoietic stem cells and a decline in the frequencies of B lymphoid progenitors with age [181,182]. In aged mice, the proliferation and differentiation of pro-B cells into pre-B cells have also been shown to be attenuated, as a result of impaired IL-7 receptor signalling, and reduced expression of surrogate light chain and RAG proteins, which are involved in the V(D)J recombination process [183], [184], [185]. Age-related alterations in IGF-1 signalling have also been characterized in pro-B cells and pre-B cells from aged mice, which may contribute to their impaired development and reduced numbers [186]. These age-related changes lead to reduced transit through the developmental stages, and eventually fewer B cell progenitors exiting the bone marrow, which can be associated with an age-related decline in the frequencies of mature follicular B cells in the spleens of aged mice [187]. This likely causes the reduced numbers of naïve B cells available for activation and entry into the GC reaction, especially during infection or vaccination with newly encountered antigens.

There are also changes in the composition of B cell subsets with age. A decrease in the percentage and absolute number of memory B cells and a corresponding increase in the percentage, but not number, of naïve B cells in the peripheral blood of older individuals have been reported [188], [189], [190]. A closer look into the different subsets of memory B cells showed that the total switched memory B cells (IgG+/IgA+, CD27+), but not unswitched IgM+ memory, are reduced in both number and percentage with age [188,191]. In addition, ageing is associated with the accumulation of a distinct subset of CD21− T-bet+ CD11c+ B cells, known as atypical or age-associated B cells (ABCs) [192]. These ABCs are also observed in high frequencies in conditions involving chronic antigen stimulation, such as HIV or malaria infection, and in autoimmune disorders, where they display a phenotype similar to anergic or exhausted cells [193], [194], [195]. ABCs upregulate inhibitory receptors, including Fc receptor-like 5 (FCRL5) and are largely refractory to BCR stimuli alone [193,[195], [196], [197]]. Instead, they proliferate extensively upon stimulation of their Toll-like receptors, TLR7 and TLR9 [191,194]. While the exact role of ABCs in the age-related decline in immune function remains to be fully characterized, it has been reported that ABCs have the potential to inhibit pro-B cell growth via TNFα-mediated apoptosis [198]. This suggests that ABCs can contribute to reduced production of mature B cells by directly inducing apoptosis and/or by exacerbating inflammaging which can have a negative impact on B lymphopoiesis [199,200].

The majority of early work on ABCs involved studying their role in chronic infection or autoimmunity, whilst their role in vaccine responses and subsequent infection is less clear. Emerging evidence has shown that ABCs are part of the normal memory B cell response that forms in response to vaccination, and are phenotypically distinct from classical memory B cells [201,202]. It has been proposed that ABCs represent a heterogenous population that can include plasma cell precursors, recently activated cells or recirculating memory B cells depending on context [202], [203], [204], [205]. After seasonal influenza vaccination of younger and older people, the formation of haemagglutinin-specific memory B cells, including FCLR5+ atypical B cells, is impaired in older individuals [79]. This is despite the accumulation of ABCs with age, and thus it is likely that the increased frequency of ABCs with age represents the memory B cell pool acquired over the multiple immune challenges throughout the individual's lifetime, and that the formation of new vaccine-responsive ABCs is impaired during ageing. As the antigen-responsive B cell pool of older people becomes increasingly dominated by ABCs with age, further investigation of how ABCs modulate vaccine-induced responses and whether they contribute to protective immunity upon (re)infection will be crucial in our attempt to understand the age-related decline in vaccine responses and how to reverse it.

The BCR repertoire diversity of both naïve and antigen-experienced B cells in older mice and humans has been shown to significantly decrease relative to their younger counterparts, based on studies that investigated the complementarity determining 3 region (CDR3) spectratype of B cells, which encode the antigen-binding regions of antibodies [206], [207], [208], [209]. This change is proposed to be a result of a decrease in bone marrow output of new B cells and increased clonality amongst antigen-experienced cells [210]. A diminished B cell repertoire diversity in older individuals might translate to reduced substrate for mounting robust antibody responses to novel antigens and reduced ability to respond to antigenically drifted epitopes, as a large diversity of B cell clones may be beneficial in the generation of diverse, protective antibodies against highly evolving viruses like influenza and HIV [211,212]. A loss of repertoire diversity in the CDR3 region of the IgM immunoglobulin genes in older individuals pre-vaccination has also been correlated with poor IgM antibody responses against pneumococcal vaccine, which might in turn translate to poorer reactivity against pneumococci [213,214]. Overall, the aged B cell compartment is characterised by high clonality and reduced diversity, and these are likely driven by the immune history of the individual during their lifetime. However, a causal link between reduced BCR repertoire diversity and defects in the vaccine-induced antibody response has yet to be fully established.

In addition to reduced antibody repertoire diversity, the selection and expansion of clones with smaller CDR3 regions post-vaccination is shown to be attenuated in older people compared to young individuals, as evident from a higher mean CDR3 size in B cells from older people after vaccination [213,215]. The expansion of immunoglobulin heavy chain genes with smaller CDR3 regions occurs after vaccination, where antibody genes with shorter CDR3 regions are selected and expanded in antigen-experienced memory cells and GC-derived cells [216,217]. As such, the higher mean CDR3 size amongst B cells from older humans post-vaccination may suggest defects in the selection process within the GC that might contribute to reduced antigen recognition strength [218].

The ability of B cells from aged mice to be activated by antigen stimulation and to participate in the GC reaction do not seem to be affected by age, when aged B cells are transferred into young mice [61,219]. B cells from aged mice and humans also do not show any functional defects in upregulating costimulatory molecules, or in differentiating into plasma cells post-stimulation [219,220]. However, several defects in B cell function that can contribute to the age-related decline in antibody response have been characterized. In particular, ageing has been associated with defects in two mechanisms that are important for B cells’ antibody diversification and affinity maturation: class-switch recombination and SHM. While class-switch recombination was long thought to occur within the GC, a recent study has confirmed that it largely occurs during the stage of the initial T cell-B cell interactions at the T:B border, prior to GC induction or extrafollicular B cell differentiation [129]. Class-switch recombination involves the recombination between two different switch regions located upstream from the heavy chain constant region, which determines the antibody's effector function [221]. This process is important in driving the production of antibodies of different effector functions and therefore is a crucial determinant of the quality of the humoral immune response. Studies involving in vitro stimulation of class-switch recombination with CD40L and IL-4 in splenic B cells from aged mice and peripheral blood B cells from older people have shown that class-switch recombination is impaired with age [188,222]. This is linked to a decrease in the expression of the AID enzyme, which is involved in the initiation of the class-switch recombination process, and its positive regulator, the E47 transcription factor [188,222]. Furthermore, the lower expression of AID in B cells from older people has been correlated with a decline in serum hemagglutination inhibition after seasonal influenza vaccination, which measures the virus-neutralizing function of protective antibodies in the serum [223]. Reduced class switching to IgG has also been observed in both plasma cells and GC B cells after a single vaccine dose in aged mice, but interestingly, this defect was no longer observed following a booster immunisation one month later [59]. It is however important to note that other studies have shown that transgenic B cells from aged mice do not show any defects in class-switch recombination, compared to B cells from young donor mice, when transferred into young hosts, suggesting that there are no cell-intrinsic defects in undergoing class-switch recombination by B cells from aged mice [219]. This may suggest some differences in B cell responses in vitro and in vivo, and/or that there are some mechanisms in vivo that can compensate for the reduced expression of AID in aged B cells. Additionally, impaired class-switch recombination in ageing might be driven by B cell-extrinsic factors, such as reduced provision of signals by T cells or a delayed initiation of the immune response upon vaccination in aged mice.

SHM occurs in the GC and involves the introduction of random mutations into immunoglobulin variable genes and their flanking heavy and light regions, a process initiated by the enzyme AID [224]. Because of its ability to diversify the BCRs in response to vaccination, SHM is important for expanding the antibody repertoire to enhance protection against a wide diversity of antigens that the organism can encounter during life [225]. Sequencing of the heavy chain variable domain of immunoglobulin (IgVH) genes of B cells microdissected from GCs in the spleen and Peyer's patch of young and old humans revealed that there was no difference in the rate of SHM between young and old donors [226]. Despite this, in the context of influenza immunisation, it has been shown that ageing is associated with a significant reduction in the accumulation of de novo somatic mutations in the IgVH genes of older people [209,227]. This may be caused by an increased reliance on existing memory B cells, which require fewer mutations, or a smaller GC reaction in older people. This reduction in intraclonal diversification results in a poorer quality of vaccine-induced antibody response, characterised by antibodies specific for highly conserved but less potent epitopes [227]. As such, older people may be less able to adapt their antibody responses to antigenically distinct epitopes on emerging drifted and shifted strains, rendering them more vulnerable to new strains of influenza virus. The process of GC B cell selection after SHM also seems to be perturbed with age, as evident from a decrease in the replacement/silent ratios in the IgVH genes in activated B cells after influenza vaccination and in GC B cells in the Peyer's patch [79,209,226]. This may suggest that apart from B cell-intrinsic defects, extrinsic changes with age, such as those described above in FDCs and Tfh cells, may also contribute to poorer affinity maturation observed in immune responses in aged individuals post-vaccination.

Together, the data suggest a minimal contribution of cell-intrinsic defects in B cells with age in mounting a GC response and differentiating into plasma cells. Rather, the age-related decline in the humoral response is likely driven by imbalances in B cell homoeostasis, marked by an accumulation of antigen-experienced B cells, and B-cell extrinsic factors that result in poorer affinity maturation of B cells and hence a decline in the quality of vaccine-induced antibody responses (Fig 1).

Conclusion

Ageing has been associated with poor vaccine efficacy, characterized by a reduction in vaccine-induced antibody responses, as most recently exemplified in vaccines against SARS-CoV-2 [14], [15], [16]. A main contributing factor to the age-related decline in antibody production after vaccination is likely a defective GC response, as the extrafollicular plasma cell response is intact [59]. Various mechanisms underlying an impaired GC response have been characterized and summarized in this review, with key driving forces likely attributed to defects in T cell function and the aged microenvironment [58,62,82,151]. While much work has gone into unravelling the mechanisms underlying the age-related decline in vaccine responses, there still remains some questions to be addressed. Much of our current understanding comes from studies involving animal models, in particular inbred mice. Whilst there are many similarities in the immune responses mounted by mice and humans, there are differences that can impede translating key findings from murine studies into a clinical setting. Non-human primates represent the closest available animal model to humans and studies involving these models have been beneficial in modelling human GC responses and age-related changes in immune function, and providing insights for clinical trial designs [229], [230], [231], [232], [233]. For direct understanding of human immune responses however, high-throughput “omics” technologies and the use of human tissue sampling have and will be essential tools in extending our understanding of how ageing modulates the immune response during vaccination in humans, alongside human challenge trials [234]. For example, ultrasound-guided fine needle aspiration of draining lymph nodes from people post-vaccination has allowed an accurate analysis of the cellular representation of lymph nodes, including GC B cells, to assess the human GC response post-vaccination [235,236]. Other factors that further add to the complexity of the question include underlying diseases, genetics and biological sex. Indeed, women have been observed to respond better than men to vaccines even during ageing [237,238]. As such, biological sex can potentially be an important variable to consider when designing strategies to improve vaccine response in older individuals. Nevertheless, current research has provided some important insights into possible strategies that can be employed to improve vaccine efficacy amongst older people. Some examples include the transient blockade of IL-10 receptor signalling to improve Tfh-dependent GC response and the use of TLR7 agonist to reverse age-related defects in cDC2 priming of T cells [58,137]. This suggests that some of the age-related changes in the immune system are not irreversible and that the ability to identify and reverse these specific pathways can potentially improve vaccine efficacy and extend the healthy lifespan amongst older people.