Age-related changes in B cells relevant to vaccine
responses
Short title: Age-related changes in B cells
Deborah Dunn-Walters, Alexander Stewart, Emma Sinclair, Ilaria
Serangeli
Affiliation: University of Surrey, School of Biosciences and Medicine
Correspondence to:
Deborah Dunn-Walters
Professor of Immunology
Faculty of Health & Medical Sciences
University of Surrey
Guildford
GU2 7XH
Tel: +44(0) 1483 68 2564
Abstract:
Older people have reduced immune responses to infection and
vaccination. B cell activation is key for the efficacy of the vaccine
response, but there are several age-related changes in B cells which
may contribute to the loss of vaccine efficacy. Different
subpopulations of B cells contain have different functions and
phenotypes. These populations can change as we age; older people
have been shown to have fewer “IgM memory” cells, regulatory B
cells and plasma cells and more IgD-CD27- “double negative” and
“Age-related B cells”. While the overall quantity of antibody in the
blood does not change, the quality of the B cell response changes;
producing less specific antibodies upon challenge and more
autoreactive antibodies. This could be due to changes in selection
pressures, as has been demonstrated by repertoire sequencing of
different subsets of B cells at different ages. Other changes in
antibody repertoire are seen, including: greater levels of IgG2 in
older people, and altered IgG1 IGHV gene usage. Since B cells rely on
their environment for efficient responses, some of these changes
may be due to age-related changes in accessory cells/signals. Other
changes appear to be intrinsic to older/aged B cells themselves,
such as their tendency to produce greater levels of inflammatory
cytokines.
Introduction
Older people have a poor response to infection, and therefore
various vaccination regimens exist to help protect them from
diseases such as influenza and pneumococcal disease [1]; reviewed
in detail in later chapters. In the same way that an older immune
system cannot respond well to infection, neither can it respond well
to vaccines designed originally for younger adults and children [2].
The immune system is complex, and so is the manner of its ageing
(immune senescence). It should be noted that immune senescence
is not synonymous with immunodeficiency, as there is no
overarching loss of function. As detailed in the previous chapters,
some elements of the immune system are preserved (e.g. number of
tissue-resident macrophages), while others are increased (e.g.
innate/inflammatory cytokine production). Increasing levels of
inflammatory cytokines with age can be a result of immune action,
cellular stress/damage, chronic diseases or increased senescent cells
in tissues secreting inflammatory cytokines as part of their
senescence associated secretory phenotype (SASP) [3]. This basal
chronic low-grade inflammation has been termed “inflammaging”
and affects immune system homeostasis. As an example, aged B
cells contribute to inflammaging by secreting increased levels of
TNF-α and IL-6 and exhibit an inverse relationship between these
levels and their response to antigenic stimulation [4, 5].
Assessments of the success of a vaccine usually depend on
measuring antibody efficiency after immunization. This may be by
measuring the titre of antibody, usually IgG, specific to the
immunising antigen by methods such as enzyme linked
immunosorbent assay (ELISA). Functional correlates of protection
are also used, for example, serum response in a haemagglutinin
inhibition (influenza [6]) or opsonophagocytic (Streptococcus
pneumoniae [7]) assay. Despite this reliance on antibody quantity
and quality as correlates of vaccine protection, we still do not fully
understand the changes in B cell development and response with
age. Nor do we fully understand the various functions of different
classes of antibody in human.
In common with many other observations in immune senescence,
there does not appear to be an age-related reduction in antibody or
B cell numbers in healthy older people, at least not in the blood [8].
In humans there have been a small number of studies looking at B
cell numbers in the follicles of tonsils, spleen and Peyer’s patches
and these do not appear to change with age either [9, 10]. In
vaccine studies, however, it has been shown that specific antibody
titres are lower and autoreactive antibodies higher in older people.
Hence much of the research into the ageing of B cells has been to
explore why the quality of the B cell response changes with age. In
undertaking these studies, using vaccination challenge to observe
the human immune system in action, novel aspects of humoral
immunity have been discovered that challenge paradigms of
immunity at all ages. Unless stated otherwise, we refer to work on
human B cells throughout this chapter and we discuss cell subsets
defined by their expression of IgD and CD27. Some work,
particularly on regulatory B cells, uses CD24 and CD38 to define cell
populations. The relationships between CD24/38 versus IgD/CD27-
defined populations has been reviewed previously [11].
An overview of B cell development
B cells can be classified into several different subsets depending on
their phenotype. These different subsets represent different stages
of lymphocyte development and/or reflect their different roles
within the immune system (Figure 1). There are also genetic
differences in the immunoglobulin loci between individual B cells
within a subset, since a population of B cells contains a large
diversity of antigen binding specificities to recognise the myriad
different possible antigens that could pose a challenge. Antigen
binding specificities depend on the B cell immunoglobulin structure.
This immunoglobulin can exist as a B cell receptor (BCR) on the cell
surface, or it can be secreted as antibody.
The BCR is an immunoglobulin made up of four subunits – 2
identical heavy chains and 2 identical light chains (Figure 2). The
chains arrange to form a “Y” shape, with the two arms having two
identical specificities for antigen and known as the Variable regions,
or Fragment variable (Fv). Specific regions of the Fv known as
complementarity-determining regions (CDRs), of which there are
three, are highly variable and therefore crucial for antigen binding.
The immense diversity of these Fv regions, estimated to be over one
billion specificities in any individual, is achieved by the process of
V(D)J gene rearrangement (Figure 3). In V(D)J gene rearrangement a
heavy chain Fv is formed from one each of three germline genes:
Variable (IGHV), Diversity (IGHD) and Joining (IGHJ) genes. There are
estimated to be 38-46 functional V genes depending on haplotype,
23 D and 6 J heavy gene segments in humans. DNA segments are cut
and re-joined, and nucleotides are removed by an exonuclease, in
addition to N nucleotides being added by the enzyme terminal
deoxynucleotidyl transferase (TdT) generating further variation. The
kappa and lambda light chains are produced using the same method
but are shorter, being made up of only kappa or lambda specific
Variable and Joining gene segments. Since these processes generate
essentially random rearrangements, in that they are not selected on
the basis of antigen recognition, they can result in a BCR that
recognises autoantigens. This potential danger to self needs to be
removed from the repertoire, so a developing B cell with
autoreactivity must either edit its BCR (by receptor editing of light
chain) or die. This process is known as central tolerance or negative
selection. Achieving an optimal balance between generation of
diverse specificities, to be prepared for immune challenge, whilst
avoiding autoreactivity is a key homeostatic challenge for the B cell
in ageing [12].
The main trunk of the immunoglobulin molecule is the constant
region, also known as the Fragment crystallizable or Fc region
(Figure 2). In a BCR this is tethered in the cell membrane and can
take various forms depending on class switching events (see later).
In a secreted antibody, other cells have Fc receptors that recognise
the constant region to carry out functions triggered by antibody.
Phagocytic cells, for example, have Fc receptors that recognise and
bind the constant region of the antibody to initiate phagocytosis of
the foreign body-antibody complex. These effector functions are
dependent on the class or subclass of the antibody (IgM, IgD, IgG3,
IgG1, IgA1, IgG2, IgG4, IgE, IgA2), which is encoded by heavy chain
constant genes (Figure 3). The central development of the B cell and
its specific BCR takes place within the bone marrow and the
resulting output of naïve B cells only express IgM and IgD on their
cell surface. Class switching to enable expression of other antibody
classes takes place during peripheral B cell maturation.
Peripheral maturation of B cells occurs after activation by antigen
and the context of activation, which can be T-dependent or T-
independent, will determine the developmental path of the B cell.
T-independent activation, which may involve stimulation of pattern
recognition receptors as well as the BCR [13], can lead to
differentiation into short-lived plasmablasts which then can secrete
antibodies such as IgG2. T-dependent activation involves the
synergistic co-stimulation of a B and T cell pair that recognises the
same antigen. This reinforcement of antigen recognition enables a
more effective response where the BCR undergoes affinity
maturation (see below) to produce an antibody with increased
affinity for its antigen. The output of this process is usually long-lived
plasma cells secreting antibodies such as IgG1.
Expansion and contraction of B cell repertoire in response to
vaccination
A key feature of the adaptive immune system is that the
lymphocytes that are selected by their antigenic specificity are
expanded. The proliferation of B cells can be followed by counting
the numbers of cells in the blood, with the peak of the post-vaccine
plasmablast response after 6-8 days [14, 15]. Despite some
successes [16], a consistent method of staining B cells to identify
antigen-specific cells is yet to be found, so qualitative methods of
measuring B cell expansion are limited. Sequencing the repertoire
of immunoglobulin genes from PBMCs has the advantage that it can
be performed on small samples and can give indirect qualitative
information on the response, as well as an indication of the change
in diversity of the repertoire during a response. There are some
inter-individual differences in gene usage. For example, CDR3
characteristics most likely reflect the effect of germline-encoded
factors related to the mechanisms of gene rearrangement [14, 15],
but it is still possible to see age and pathology-related changes in
repertoire beyond these background differences. Older people
often have reduced B cell diversity at baseline which correlates with
poor health [14]. Repertoire analyses have shown us that a response
to a vaccine challenge is not dominated by a single BCR but consists
of a diverse collection of B cells expanding together. The responding
repertoire in older people has been shown to be less diverse and the
magnitude of expanding clones is reduced compared to that of
younger subjects, particularly in IgA [17, 18].
Looking for differences in individual immunoglobulin genes in
healthy people responding to challenge has proven to be less
discriminatory than was hoped, since it is difficult to identify V gene
use that differs with age. The CDR3 region however, at the V(D)J
gene junction, appears to be an excellent indicator of repertoire
selection. Looking in total PBMCs, vaccination results in smaller, less
hydrophobic CDR3 regions in all classes of antibody at day 7 after
challenge, returning to baseline values at day 28, leading one to
assume that small hydrophilic CDR3 regions are desirable for
efficient immune responses. These results likely reflect the character
of the plasmablast response, which peaks around day 7 after
challenge. It is therefore significant to note that older subjects did
not show the same level of CDR3 selection in response to vaccine.
Moreover, they appeared to have longer, more hydrophobic CDR3
regions even in the unmutated IgM population prior to any
challenge, which would put them at a disadvantage in the selection
of short hydrophilic sequences in response to vaccine. Longer,
hydrophobic, CDR3 regions have been associated with increased risk
of autoreactivity [19]. Later work (see below) showed that this
difference in the naïve population was likely the effect of poor
negative selection in early B cell development [20] and reminds us
that the observed character of repertoire is the cumulative effect of
both positive selection for exogenous antigen specificities and
negative selection of cells recognizing autoantigens. Studies in mice
have shown that auto-reactive antibodies can arise both from
germline genes, and from antigen-experienced mutated genes [21],
so the process of negative selection occurs at all stages of B cell
development.
Early B cell development in the bone marrow
A healthy and diverse B cell population requires efficient early
development and the bone marrow environment is critical for this.
The composition of the bone marrow is complex and can be affected
by peripheral antigen challenge, since haematopoietic stem cells can
respond directly to infection via TLRs, or indirectly by sensing a
change in cytokine environment [22]. It undergoes substantial age-
related changes, such as decreased cellularity and increased fatty
tissue, which would likely have a significant effect on
haematopoiesis. Data from mice indicate that there are age-related
epigenetic changes even at the haematopoietic stem cell stage [23]
and that B cell formation is reduced, with fewer cells undergoing
efficient gene rearrangement to make it to the pre-B cell stage of
development [24]. Recent evidence has suggested that
accumulations of age-associated B cells (see below) and the
resulting inflammatory environment may cause increased cell death
at the pro-B stage in aged mice [25]. Despite the reduction in B cell
generation, the numbers are still sufficiently high throughout most
of life such that we remain numerically B cell sufficient into very old
age. In normal circumstances, many B cells are lost as they do not
pass through the tolerance checkpoints [26]. However, a reduction
in the rate of B cell output, or a change in environmental signals,
may alter these tolerance checkpoints and thereby the quality of the
B cell repertoire that emerges into the periphery; either in terms of
increased auto-reactivity or reduced affinity for antigen. Indeed, it
has been shown that changes in characteristics of the
immunoglobulin genes that normally occur in the repertoire of
young people as the cells develop from preB cells to naïve B cells,
are reduced in older people [20]. In particular, the size of the CDR3
region of the immunoglobulin was found to be larger in immature
and naïve B cells of older people [20]. The exact meaning of this is
unclear but the CDR3 is the main part of the antibody that binds
antigen. Large CDR3 have been correlated with autoreactive
antinuclear antibodies [19] and have been shown to exhibit binding
promiscuity [27]. Therefore, a credible hypothesis is that changes in
bone marrow B cell development result in the release of increased
numbers of B cells with larger CDR3 regions that have the ability to
bind autoantigens; this could explain the observation that older
people have more autoreactive antibodies [19]. Thus, the
homeostasis of the older B cell repertoire is compromised even
before any antigen challenge from infection or vaccination.
Peripheral B cell activation and the germinal centre
In the classical T-dependent B cell reaction there is co-operation
between the two types of lymphocytes in secondary lymphoid tissue
which results in the formation of germinal centres and affinity
maturation of the B cell receptor. Affinity maturation comprises
somatic hypermutation to alter the immunoglobulin genes in B cells,
coupled with a Darwinian selection process of those B cells with the
highest antigen affinity. When a B cell receives appropriate help
from T cells it enters the germinal centre and proliferates in the dark
zone as a germinal centre centroblast. Centroblasts upregulate
telomerase to counteract the effects of multiple cell divisions on
telomere length and, in this context, it is interesting to note that
better immune responses to vaccination correlated with increased
telomere length of B cells in older people [28]. Germinal centre B
cells express Activation Induced cytidine Deaminase (AID) which
catalyses the process of somatic hypermutation of the
immunoglobulin gene. The B cells then move to the light zone as
centrocytes where rescue signals are provided by follicular dendritic
cells (FDC) and T follicular helper (Tfh) cells. Since germinal centre
centrocytes do not have the anti-apoptotic protein Bcl2, they will
die by apoptosis unless they receive rescue signals which are
conditional on efficient recognition of the antigen by the newly
formed B cell receptor. As FDC and Tfh are limited in number, only
the B cells with the highest affinity BCR will be selected. With T cell
help the resulting B cells can also switch the class of their antibody
from IgM to IgG/IgA/IgE. The germinal centre is therefore a crucial
microenvironment for the production of high affinity class switched
immunoglobulin in response to infection or vaccination and has
been highly studied.
Histological studies of human tissue did not see any significant age-
related differences in the size of germinal centres in tonsils, spleen
or Peyer’s patches, but some differences in T cell populations were
noted [10, 29]. With regards to the quality of the germinal centre
response, there have been reports of intrinsic B cell changes in
development, namely that the expression of AID is reduced in older
adults. Transcription of AID is controlled by a tyrosine kinase
cascade that activates the transcription factor E47, and E47 mRNA
stability has been shown to be decreased in old B cells due to
decreased phospho-MAPKinase and phospho-TTP (tristetraprolin)
[30]. Post-transcriptional decrease of AID and E47 has also been
shown to be mediated by the miRNAs miR-16 and miR-155 in older
adults [31]. Other studies, of the lineages of B cells in human
germinal centres have indicated that the effects of age on the
number of mutations occurring in individual germinal centres is
minimal in comparison to the age-related effects on selection of
cells [32]. A decrease in the strength of selection in the germinal
centre would result in reduced affinity for antigen and thereby a
reduced ability to combat challenge. Since the selection process
involves B cell interactions with other cells, it is clear the availability
of appropriate help in the extrinsic cellular environment is also
critical for production of high affinity antibody. The products of the
germinal centre are either memory cells, which retain the capacity
to re-enter the germinal centre in response to challenge at a later
date, or plasmablasts which eventually mature into plasma cells
secreting large quantities of antibody. Plasma blasts can also be
generated in a T-independent manner from pre-existing memory
cells in an extrafollicular response.
Plasma cells
Sufficient T cell help will result in upregulation of the master
regulator of plasma cell differentiation, transcription factor Blimp1
[33]. Without such help, surviving B cells are more likely to have
higher levels of the transcription factor Bach2 and differentiate into
memory cells [34]. It has been shown that B cells in older people
have decreased Blimp-1 [35]. This would result in decreased plasma
cell output and indeed it has been reported that plasma cells are
less prevalent in older people in the peripheral blood [36, 37] and in
the bone marrow where these types of cells are mainly found [38].
Another indication that the numbers of plasmablasts are reduced
comes from repertoire studies, where the immunoglobulin
repertoire of peripheral blood has shown a marked decrease in the
prevalence of sequence “clones” in older people 7 days after an
influenza vaccination [17].
Monoclonal gammopathy of undetermined significance (MGUS) is
an asymptomatic plasma cell pathology that is present in more than
3% of the over 50’s and has an average multiple myeloma
progression risk of 1% per year [39]. This arises from a clonal
expansion of plasma cells in the bone marrow secreting monoclonal
(M) paraprotein and is associated with increased susceptibility to
infections. Abnormal clonal expansions of plasma cells in the bone
marrow niches will upset the homeostasis of the older immune
system even further, therefore it is perhaps not surprising that a
reduced vaccination response has been also been reported in
patients with MGUS [40].
Memory B cells
Cells that leave the germinal centre as Memory B cells express high
levels of the anti-apoptotic Bcl-2, which contributes to their long-
term survival. They may also express activation markers such as
CD27. Classical memory cells no longer express IgD and can either
still be expressing IgM or have undergone class switching, therefore
they are identified as CD19+IgD-CD27+. This subset of cells
generally does not change in number with age although there are
qualitative differences in the repertoire.
Memory cells carry the evidence of their antigen experience in their
immunoglobulin genes. Mutations in memory cell variable regions
reflect somatic hypermutation and changes in the constant region
are the result of class switch recombination. Since IgG is the most
common antibody in circulation it has been the most widely used as
a correlate of protection [17] and distinguishing between different
subclasses of IgG has not previously been a priority. This is despite
the fact that there are significant functional differences between
IgG1/IgG3 versus IgG2 due to differences in the binding affinities of
their constant regions[41] and IgG2 is more often produced in a T-
independent response [41]. With the advent of long read repertoire
analysis it has been possible to see that memory B cells of older
people have more IgG2 than IgG1 cells both at baseline and in
response to vaccine, perhaps indicating a lack of T cell help [12, 17].
The balance of selective forces acting on the two different IgG
responses result in a different repertoire for IgG1 than for IgG2, but
in older memory cells the IgG1 loses its distinctive repertoire, again
indicating that repertoire selection is compromised in older age [42].
IgM memory B cells
The origin of IgM memory cells may be from the germinal centre
reaction or from extra-follicular B cell activation. These cells in the
peripheral blood retain their IgM and IgD expression but also have
mutations in their immunoglobulin genes and express CD27 [43].
There is a similar group of cells in the marginal zone of the spleen
[44], although later detailed repertoire analysis indicated there may
be different functions between the two [45]. The number of IgM
memory cells in the blood are known to diminish with age [30]. The
exact function of IgM memory cells is controversial but since this
population has a different immunoglobulin repertoire from that of
IgA, IgG1, IgG3 populations we can assume that different selective
pressures have acted on these cells during their development [46]. It
is also thought that IgM memory cells are a critical component of
antibacterial immunity, such as the T-independent response to
pneumococcal polysaccharide, which is of extreme importance in an
elderly population susceptible to bacterial infection [19, 42, 47, 48].
There is also evidence that IgM memory cells are better than class
switched memory cells at carrying immune memory out of germinal
centres in a T-dependent response, so this population may have
multiple origins and functions. A high dimensional phenotypic
analysis showed that the relative levels of IgM and IgD on the cell
surface can be used to distinguish different subpopulations within
the IgM memory pool. In particular, IgMHiIgDMedCD27+ cells are
increased in the aged at the expense of the IgMMedIgDHiCD27+ cells
[42]. Further investigation to determine the functional
consequences of these changes for older people is required.
“Double Negative” Memory B cells
In common with classical memory cells, this population contains
cells that do not express IgD (although they may express IgM) and
have mutated immunoglobulin genes, however, it lacks CD27
expression. As such these cells have been named “Double Negative”
(DN) cells but have also been referred to as “atypical memory B
cells”, largely because their developmental pathway and their role
has yet to be fully elucidated. The absence of CD27 has led to
speculation that these cells are exhausted and have therefore
downregulated their activation molecules [49]. In comparison to
classical memory cells, however, the overall level of repertoire
mutation in DN cells is lower which could imply they have been
exposed to fewer activation stimuli than their younger counterparts.
Double negative cells have been shown to secrete granzyme B upon
stimulation and have a tissue-homing phenotype [50]. Other studies
suggest that these cells may have arisen from T-independent
stimulation since they are present in individuals that are CD40
deficient (a key co-stimulatory molecule on T helper cells). Another
hypothesis is that a large majority of these cells are classical
memory cells that have lost their activation markers through
absence of sufficient secondary stimulatory signals. In an effort to
elucidate the relationship between these DN cells and CD27+
classical memory cells the immunoglobulin repertoires of the two
subsets were compared and very little difference was found
between them [51]. In some instances, there were examples of the
same clonal family of cells split between the two populations, but it
was not possible to infer consistent parental relationships. The
potential role of these cells is important since there is clear and
consistent evidence that this population increases with age [4, 5].
Double negative cells have also been shown to be increased in
patients with autoimmune diseases, such as systemic lupus
erythematosus (SLE), [52] and chronic infectious diseases [53]. In
view of the evidence of multiple phenotypes and functions it is likely
that this subset of cells is made up of a number of different cell
types and may also contain the human equivalent of the mouse
“Age-related B cells” (ABCs) [54].
Age-associated B cells
Age-associated B cells were originally identified in mice as a type of
memory cell that increases with age [54, 55], is found in blood,
spleen and bone marrow, but is rarely observed in lymph nodes
[56]. ABCs are characterized by a T-bet driven transcriptional
program and are CD21 negative [54]. They appear to be controlled
by a signalling pathway involving IL21, SWAP-70, DEF6 and IRF5
[57]. Several studies have established that ABCs are effective at
presenting antigen both in vitro and in vivo [56]. When activated by
either TLR7 or TLR9 agonists, ABC secrete a variety of cytokines
including IFNγ, IL-4, IL-6 and IL-10. Upon activation, ABCs rapidly
differentiate to antibody secreting plasma cells and then undergo
class-switching toward IgG2a/c [56]. Beyond their age related
accumulation, these cells seem to play a role in both normal and
pathogenic responses at all ages [56]. A large majority of mouse
ABCs express memory markers and have mutated immunoglobulin
genes [58].
Since the discovery of ABCs in mice it has been shown that
Tbet+CD21- B cells can also be found in humans, they increase with
age and their presence correlates with poor vaccine response [59].
They have also been shown to be more prevalent in autoimmune
diseases such as systemic lupus erythematosus (SLE), rheumatoid
arthritis [52, 57], and in chronic infection in both mice and humans
[60]. Although these cells have been connected with autoimmune
disease and ageing it is still unclear what function these cells have in
the wider immune system. It is likely that there is a large degree of
overlap between the double negative memory B cell population
defined as CD19+CD27-IgD- and the ABC population defined as
CD19+CD21LowTbet+.
Regulatory B cells
Regulatory B cells (Bregs) support immunological tolerance by
suppressing the functions of other immune cells such as CD4+ Th1
cells, CD8+ cytotoxic cells, dendritic cells and monocytes. They can
also affect the function of T regulatory cells and iNKT cells [61].
Many different B cells have been shown to produce the inhibitory
cytokine IL10 upon appropriate stimulation, and IL10-secreting B
cells have been shown to decrease with age [62]. However, many
different types of B cells can secrete IL10 upon stimulation. One
particular group of cells has been shown to contain potent
suppressors of the immune response, both by secretion of IL10 and
by other contact-dependent mechanisms and is defective in
autoimmune disease [63]. These cells are CD19+CD24HiCD38Hi and
are not class switched, so they retain IgM and IgD. The majority are
thought to be mutation-free, although some cells in this group may
be CD27+ and so would fall into the “IgM memory” population of
cells carrying mutations in their immunoglobulin genes. Current
thinking is that, after excluding IgM memory cells by inclusion of the
CD27 marker, it is these CD19+CD24HiCD38HiCD27- cells that are the
true regulatory B cells. These have been shown to be decreased
with age [37] [62] .
Extrinsic environmental influences on B cell function
The most well-known examples of B cell microenvironments that
affect function are the bone marrow and the germinal centre, as
mentioned above. In addition to the role of the bone marrow in
supporting haematopoiesis, it also provides survival niches for
plasma cells and memory T and B cells [64]. Tantalising glimpses of
unusual cell interactions, such as plasma cells with eosinophils [65]
or regulatory T cells [66] are emerging which indicate we have much
to learn about human bone marrow function. As previously
mentioned, we know much more about the interaction of B cells
with T cells in the germinal centre via CD40/CD40L signalling. It is
interesting to note that B cells, as antigen presenting cells (APCs),
play a role in regulating T cell tolerance in the thymic
microenvironment and this role appears to decrease with age [67].
It is also becoming clear that B cells carry pattern recognition
receptors (PRR) and can respond to antigens without the aid of Tfh
cells. They can also receive stimuli from many different types of
cells such as natural killer (NK) cells, CD1d-restricted invariant
natural killer T (iNKT), NKT-like lymphocytes [68, 69] and mucosal-
associated invariant T cells (MAIT). These unconventional helper
cells also show age-related differences that may affect overall B cell
function: CD1d-restricted invariant natural killer T (iNKT) cells are
innate-like T lymphocytes that rapidly respond to stimulation with
specific lipid antigens that are derived from infectious pathogens or
stressed host cells. Human peripheral blood iNKT cells decrease in
number, and change in cytokine profile with age [70]. NKT-like
lymphocytes are a subset of T cells that show a highly specialized
effector memory phenotype, and can be increased with age [68, 70].
Mucosal-associated invariant T cells are innate-like T cells
comprising up to 10% of the peripheral blood T cells in humans but
are enriched in the mucosal sites and have been shown to be able to
activate B cells [71, 72]. Levels of MAIT increase up to the fourth
decade in humans and then dramatically decline [72], with older
MAIT cells showing an increase in IL-4 secretion [73].
Cautionary notes
There may be many confounding factors affecting B cells in the
immune system that are only recently emerging and therefore
would not have been taken into consideration at the time of study,
in the studies discussed here. In chronobiology for example, the
time of day that a vaccine is administered can affect the measured
response [74]. Furthermore, older people have been shown to have
different baseline antibody titres depending upon the times of day
that samples were taken, and this can lead to misleading post-
vaccine study outcomes [75]. The same study noted that patients
taking medications, a common occurrence in elderly people, such as
NSAIDS, metformin or statins, have altered B cell and antibody
responses to vaccination [76]. Subclinical chronic infections, such as
Cytomegalovirus and Epstein Barr virus, have been shown to affect
the B cell repertoire regardless of the age of the individual, although
this may not have relevance to the ability of an individual to respond
to vaccine [77]. Another possible confounder is the metabolic status
of the individuals. There are more overweight and obese people at
older ages [78, 79] and a rise in adipose tissue increases levels of
inflammatory cytokines and promotes the recruitment of immune
cells resulting in low-grade inflammation and dysfunctional
metabolism [80]. Several reports have suggested that because
obesity affects immune function, it could lead to a poor response to
vaccines [81, 82]. There is some evidence that the local and systemic
inflammation established in the adipose tissue leads to an increase
of autoreactive IgGs [83]. In this regard it is notable that Bregs have
been associated with adipose tissue [84] and blood samples from
overweight and obese individuals have been shown to have contain
fewer Bregs compared to individuals of normal-weight [80].
Likewise, a study of mice fed a high fat diet has shown that the
repertoire of B cells is affected by the increased adiposity [85]. Thus,
in any study of B cell vaccine response it will be important pay
careful attention to selection of volunteers and to gather as much
patient information as possible in order to consider potential
confounding factors.
Summary and future directions
Despite the reliance on B cells and antibodies as a means for
measuring vaccine responses there are few studies that directly
relate the three components of age, B cells and vaccine immunity.
There are, however, many studies that look between two of these
components, most commonly age and B cells or B cells and vaccine.
As such, changes in B cells with age and their effects on vaccine
responses are mostly inferred from separate studies, as has been
apparent throughout this chapter. The vast majority of these age-
related changes involve some level of change in how B cell
development is regulated resulting in changes in cellular phenotype
proportions and antibody repertoire. Some of these changes e.g. the
presence of ABCs and longer more hydrophobic CDR3s, are
associated with diminished response to infection or vaccine. It
follows that such changes have a major impact on how the aged
respond to infection, even if the exact cause of such changes are not
fully understood. Separating age-effects on the B cell response into
extrinsic versus intrinsic factors affecting development is difficult to
assess and development of better in vitro experimental techniques
would greatly enhance our understanding. With improved
techniques and a greater understanding of B cell biology future
studies should take a more holistic approach to questions
surrounding age, B cells and vaccination.
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