Download Transcriptional networks controlling B cell germinal center activities

Transcriptional networks controlling B cell germinal center activities
Timothy Recaldin1,2 and David J. Fear1,2
Division of Asthma, Allergy and Lung Biology, King’s College London, London, United
1
Kingdom.
2
Medical Research Council & Asthma U.K. Centre in Allergic Mechanisms of Asthma,
King’s College London, London, United Kingdom.
Corresponding author: David J. Fear: Dept. Asthma, Allergy and Respiratory Science,
King’s College London, Guy’s Hospital, St. Thomas Street, London, SE19RT, United
Kingdom, [email protected]
Abstract
Diversification of the antibody repertoire is essential for the normal operation of the
vertebrate adaptive immune system. Following antigen encounter, B cells are
activated, rapidly proliferate, and undergo two diversification events; somatic
hypermutation (followed by selection), which enhances the affinity of the antibody for
its cognate antigen, and class switch recombination, which alters the effector functions
of the antibody to adapt the response to the challenge faced. B cells must then
differentiate into antibody-secreting plasma cells or long-lived memory B cells. These
activities take place in specialised immunological environments called germinal
centers, usually located in the secondary lymphoid organs. To successfully complete
the germinal center activities, a B cell adopts a transcriptional program that allows it to
migrate to specific sites within the germinal center, proliferate, modify its DNA
recombination and repair pathways, alter it apoptotic potential and finally undergo
terminal differentiation. To coordinate these processes, B cells employ a number of
“master regulator” transcription factors which mediate wholesale transcriptomic
changes. These master transcription factors are mutually antagonistic and form a
complex regulatory network to maintain distinct gene expression programs. Within this
network, multiple points of positive and negative feedback ensure the expression of the
“master regulators”, augmented by a number of “secondary” factors that reinforce
these networks and sense the progress of the immune response. In this review we will
discuss the different activities B cells must undertake to mount a successful T celldependent immune response and describe how a regulatory network of transcription
factors controls these processes.
Introduction
In order for our bodies to mount a successful humoral immune response, B cells must
first encounter, and then be activated by, their cognate antigens. Following activation,
the B cell (or its progeny) diversifies the antibody it produces, increasing its affinity for
antigen and altering its effector function, thus tailoring the response to the
immunological challenge faced. The B cell then differentiates into either a specialised
“antibody-secreting cell” (plasmablast or plasma cell (PC)) to eliminate the challenge,
or a long-lived memory B cell, so that a more rapid (and specific) response can be
mounted upon reencountering the same foe. This sequence of events takes place
within a specialised immunological environment termed the germinal center (GC). First
described by Flemming in 1884 (1), the GC is a transient structure that forms within
secondary lymphoid organs such as the lymph nodes, spleen and Peyer’s patches,
and is essential for an efficient response to a T cell-dependent antigen. GC like
structures can also form within “local” ectopic tissues(2), promoting B cell
diversification(3, 4), where they play an important role in the pathology of chronic
inflammation, infection, autoimmunity and atopic disease.
In the GC, B cells undergo rapid clonal expansion (proliferation) and express the
genome mutator enzyme activation-induced cytidine deaminase (AID). AID is
indispensible for two key GC processes, somatic hypermutation (SHM) and classswitch recombination (CSR). SHM introduces non-templated mutations in the
immunoglobulin (Ig) variable region, changing the antibody’s affinity for antigen
(reviewed in (5)). CSR alters the antibody effector functions by recombination of the
variable region (initially linked to the μ and δ heavy chain exons, encoding IgM and IgD
respectively) with one of the downstream constant region genes of the α, γ, or ε
isotypes, encoding IgA, IgG and IgE. Each antibody isotype initiates different
downstream immune reactions and thus adapts the response to the nature of the
challenge faced (6). Together these processes shape the antibody response,
producing GC B cells with high affinity, class-switched antibodies that can differentiate
into either antibody-secreting PCs or long-lived memory B cells.
Lymphocytes are the only vertebrate somatic cells that undergo such drastic and
potentially dangerous modifications of their genomic DNA as part of their “normal”
biology (including VDJ recombination that occurs during B and T cell ontogeny (7)). It is
critical that these events are tightly regulated; accumulation of dangerous “off-target”
mutations poses a significant threat to genome integrity and likely explains why B cells
are highly prone to transformation into a variety of leukemias/lymphomas(8).
Successful completion of the GC reaction therefore relies on careful regulation and
coordination of B cell movement, division, apoptosis, differentiation, DNA repair and
recombination. This is achieved through the activation (and repression) of multiple
transcriptional programs that interact in a series of complex regulatory networks,
discussed in this review.
Germinal center structure, formation and B cell movement
GCs are not permanent structures, but rather arise transiently within the lymphoid
tissue such as lymph nodes (and local tissues) in response to a T cell-dependent
antigen (Figure 1). Lymph nodes are composed of multiple lobules surrounded by
lymph-filled sinuses enclosed by a capsule (9). Naïve B and T cells from the circulation
continually cycle through the lymph node, residing within distinct areas of the lobule
where they can interact with specialised antigen presenting cells (APCs) to survey the
antigen environment. The outer layer (cortex) contains follicles of naïve B cells and
follicular dendritic cells (FDCs). These follicles are separated from the T cell zone
(paracortex), which contains naïve T cells and dendritic cells (DCs), by the
interfollicular zone (10, 11).
Circulating naïve B cells bear the chemokine receptor CXCR5 and are thus attracted to
the lymphoid follicles by the chemokine CXCL13, which is expressed by resident FDCs
and marginal reticular cells (12). Similarly, circulating naïve T cells expressing the
chemokine receptor CCR7 are recruited to the T cell zone by fibroblastic reticular cell
expression of the CCR7-ligands, CCL19 and CCL21, (13).
Once in the follicles, naïve B cells interact with antigen via their B cell receptor (BCR).
The antigen is usually displayed on the surface of FDCs, but can also be captured from
macrophages lining the subcapsular sinus (SCS) (14, 15). BCR binding to cognate
antigen activates the B cell, triggering internalisation of the BCR:antigen complex and
subsequent presentation of antigen on the cell surface in the context of MHC class II
molecules (16). BCR engagement also upregulates expression of the chemokine
receptor CCR7 which promotes B cell migration to the periphery of the T cell zone
where its ligands, CCL19 and CCL21, are abundantly expressed (17, 18). During this
phase of activation, the B cells continue to maintain expression of CXCR5; the balance
between CXCL13 expression in the follicles and CCL19/21 expression in the T cell
zone positions B cells at the border of the T cell zone (17). Meanwhile, naïve T cells in
the T cell zone encounter their cognate antigen, here presented by DCs, initiating
commitment towards a T follicular helper (TFH) cell phenotype (19). TFH cell
commitment is accompanied by CCR7 downregulation and CXCR5 upregulation,
promoting TFH migration to the T/B cell boundary where they can support B cell
expansion(20, 21).
Two days after antigen encounter, activated B cells find their cognate TFH cells and
form long-lived interactions that result in full B cell activation and proliferation(22, 23).
At this time, a subset of activated B cells move away from the extrafollicular sites into
the SCS where they differentiate into short-lived plasmablasts. These cells secrete
IgM, providing immediate (albeit low specificity) protection to the individual (24). After 3
days, the activated TFH and B cells migrate into the centre of the follicle where the B
cells start to rapidly proliferate. By this time the B cells begin to express the “master
regulator” B cell lymphoma 6 protein (BCL6, see below), which drives the acquisition of
a “GC B cell” phenotype (25). Of note, BCL6 expression is also responsible for
commitment of T cells to the TFH fate (26), thus regulating both the T and B cell GC
transcriptional programs. The rapid proliferation of activated GC B cells within a
network of FDCs, pushes aside the resident follicular B cells to form the early GC over
days 5-6. By day 7 the rapid proliferation of GC B cells, coupled with the continued
influx of activated GC cells, results in the polarisation of the fully formed GC into two
distinct microenvironments, the so called dark and light zones.
In the dark zone, densely packed GC B cells, referred to as centroblasts, divide rapidly,
and undergo SHM. Centroblasts are retained in the dark zone by their expression of
the chemokine receptor CXCR4, the ligand of which, CXCL12, is abundantly
expressed by dark zone stromal cells(27). Downregulation of CXCR4 and upregulation
of CD83 and CD86 allow the GC B cells to migrate from the dark zone into the light
zone, a less densely packed compartment populated with TFH cells, macrophages and
FDCs (28). In the light zone, B cells undergo a process of Darwinian selection whereby
B cells producing higher affinity antibodies compete for available antigen (29, 30)
and/or T cell help(31, 32), thus receiving survival signals via BCR binding. Selection
promotes centrocyte reentry into the dark zone for further rounds of mutation and
selection. (Reviewed in Victoria (33). Conversely, lower affinity B cells, receive no
survival cues and undergo apoptosis (34). In parallel, CSR drives apoptosis of
undesirable B cell clones through deletion of the Ig heavy chain in a process called
locus suicide recombination. This prevents BCR expression and thus eliminates the
survival signals the BCR transmits, inducing apoptosis (35). Having survived selection
in the light zone, GC B cells can do one of three things: They can re-enter the dark
zone for additional rounds of proliferation and SHM (36, 37). Alternatively, GC B cells
can leave the GC and differentiate into plasmablasts (precursors of antibody-secreting
PCs) or they can differentiate to form long-lived memory B cells to enable a rapid
response upon reencountering the same antigen (reviewed in (38, 39)).
Coordination of all the events described above are controlled by a number of “master
regulator” transcription factors (Figure 2). In the remainder of this review we will
discuss the role of these transcription factors and how their interacting regulatory
networks control the GC activities.
Master regulators of B cell identity
PAX5
Paired box protein 5 (PAX5) is the master regulator of B cell identity and is expressed
throughout B cell development (40), from pro-B cells (41) through to mature GC B cells
(42). PAX5 directly binds to thousands of DNA sites in B cells and functions by both
activating and repressing gene expression (40). During early B cell development PAX5
is absolutely required for the initial commitment of lymphoid progenitors to the B cell
fate (43) and V-DJ recombination of the Ig locus (44, 45). In mature B cells it regulates
the expression of genes critical to B cell identity, including components of the B cell
receptor (Ig heavy chain and CD79A), CD19, CD21, BLK, IRF4 and IRF8(40). In
addition, PAX5 further reinforces B cell identity by repressing the expression of lineage
inappropriate genes including FLT3, CCR2 and CD28, which are expressed in PCs
following PAX5 downregulation, and M-CSF receptor, NOTCH1, RAMP1, LMO2 and
CCL3, which are expressed in common lymphoid progenitors and myeloid cells (46).
As PAX5 promotes and maintains the expression of the B cell transcriptional program,
its downregulation is required for differentiation into committed Ig-secreting PCs (47)
(see master regulators of plasma cell identity). Critically, PAX5 directly represses the
expression of one of the master regulators of the PC program, XBP1, and its
downregulation is required for Ig secretion(48, 49).
BCL6
As mentioned earlier, B cell lymphoma 6 protein, BCL6, is essential for GC formation
(50) and is considered the master regulator of the GC, where it controls gene
expression programs in both GC B cells (25) and in TFH cells(26) (reviewed in Basso
and Dalla-Favera, 2012(51)). Within these cells BCL6 predominantly functions as a
transcriptional repressor, directly suppressing multiple genes involved in the DNA
damage sensing pathway, including TP53, ATR and CHEK1 and regulators of the cell
cycle, p21, p53 (52, 53); this establishes a transcriptional program that allows both the
rapid proliferation of cells and the tolerance of DNA damage essential to SHM. In
addition, BCL6 controls the migration of B cells into the follicle. To this end, BCL6deficient GC B cells fail to upregulate CXCR4 (25), the chemokine receptor responsible
for GC localisation into the dark zone, and fail to downregulate sphingosine-1
phosphate receptor type 1 (S1PR1) (54), which facilitates trafficking of B cells out of
the follicles (55). One of the critical functions of BCL6 appears to be the repression PC
differentiation, in this case mediated by repression of BLIMP1(56) (see below): Thus,
expression of BCL6 in the GC B cell not only activates GC B cell identity but
concomitantly blocks establishment of the PC program. Although BCL6 functions as a
transcriptional repressor, it also indirectly induces AID expression in GC B cells (50) by
inhibiting expression of miR-155 and miR-361, two negative regulators of AID (57).
Similarly, repression of Spi1, IRF8 and MYB is also relieved, all regulators of the GC
transcriptional program. Further supporting GC activities, BCL6 has been shown to
regulate B cell/TFH cell interactions, through an undetermined integrin dependent
mechanism(25).
BLIMP1 and XBP1: Master regulators of plasma cell identity
Two transcription factors, B lymphocyte-induced maturation protein 1 (BLIMP1, also
known as PR domain zinc finger protein 1, PRDM1) and X-box-binding protein 1
(XBP1) are essential for orchestrating PC differentiation (reviewed in (38). BLIMP1 is a
transcriptional repressor that, within the B cell lineage, is exclusively expressed in
antibody-secreting cells, being expressed at a low level in plasmablasts and high level
in mature PCs (58, 59). During PC commitment, BLIMP1 represses the expression of
the B cell specific regulators, PAX5, BCL6, ID3, cMYC and SPIB, (47, 60), thus
allowing expression of XBP1(47). However, although XBP1 appears to act downstream
of BLIMP1 in the regulatory network(61), BLIMP1 is necessary, but not sufficient for
XBP1 expression(47). Furthermore, it seems that BLIMP1 is not required for initiation
of the PC differentiation program since pre-plasma-blasts form in the absence of
BLIMP1(62).
XBP1 acts downstream of BLIMP1, and is a key regulator of PC development, but it is
not absolutely required for the formation of antibody-secreting cells (63). Rather, XBP1
appears to predominantly act to set up the cells to allow for the secretion of vast
quantities of Ig (64), inducing endoplasmic reticulum remodeling, activation of mTOR
(65) and autophagic pathways(66) and the induction of the unfolded protein
response(64). Although much is known regarding the interconnections that exist
between the regulatory networks of these B cell lineage master regulators, questions
still remain as to exactly what initiates each pathway.
Other factors implicated in regulating GC activities.
NF-B
The classical NF-B pathway consists of 3 subunits, cREL, RELA and p50, forming two
major heterodimers, cREL/p50 and RELA/p50 (67). Both cREL and RELA are induced
transiently upon BCR ligation, peaking 1-2 hours after stimulation (68). Here, they
induce expression of chemokines that promote B: T cell interaction, namely CCR7,
which is responsible for B cell movement to the periphery of the T cell zones (17), and
CCL3 and CCL4, two T cell chemoattractants (69).
In the GC, cREL and RELA have non-redundant roles. While NF-B expression is
generally absent in GC B cells, it is detected in a small subset of centrocytes in the
light zone. These cells likely represent positively-selected B cells being primed for
cyclic reentry into the dark zone. cREL-ablation in this context results in the collapse of
the GC. Unlike cREL, RELA-ablation has no impact on GC maintenance, but instead
results in a significant reduction in the prevalence of PCs and a subsequent loss in IgG
serum titres, through impaired upregulation of BLIMP1(67).
cMYC
cMYC is a global driver of cell growth and division. However, counter-intuitively, it is
actively suppressed in the rapidly dividing GC centroblasts by BCL6 (70, 71).
Therefore, dark zone GC B cells must proliferate in a MYC-independent manner. This
is, at least in part, facilitated by BCL6 interaction with the MYC-binding protein, MIZ1,
which together suppress transcription of the cell cycle arrest gene CDKN1A (72).
Despite its absence in centroblasts, cMYC still plays an essential role in the initiation
and functioning of the GC. It is transiently detected 2 hours after antigenic stimulation,
providing a boost in the population of antigen-responsive B cells prior to GC
commitment. Accordingly, cMYC ablation prior to immunisation prevents the
development of the GC (73).
cMYC is also detected in a subset of B cells localised to the light zone and is likely
involved in their re-entry into the dark zone. Consequently, GC B cells that have been
engineered to lose cMYC expression after GC formation, display a collapsed GC
response. The purpose of this transient, yet indispensible, bout of cMYC expression is
not clear but may represent a mechanism by which selected B cells are primed for
another wave of proliferative expansion in the dark zone (70).
IRF4
IRF4 is a member of the IRF (interferon regulatory factor) superfamily of transcription
factors that shows relatively weak DNA binding on its own. Therefore, in order to exert
its diverse functions it binds DNA cooperatively with a host of other transcription
factors, including IRF8, PU.1 and SPIB (74, 75). IRF4 plays an essential role in isotype
switching, with IRF4-deficient mice failing to induce AID expression and undergo CSR
when stimulated in vitro (76, 77). IRF4 may regulate AID expression through
cooperative binding with BATF, a transcription factor essential for AID expression(78),
through binding to AP-1-IRF composite element motifs (79).
IRF4 is rapidly induced upon BCR ligation (79, 80) and reported to be required for
BCL6-induction and entry into the GC reaction. However, it is not required for
maintenance of the GC as deletion at a later points does not impair the GC reaction
(76). In addition to establishing the GC reaction, IRF4-deficient mice also fail to make
mature PCs (76, 77). Sciammas et al(77) report that this defect is a result of failure to
induce BLIMP1 expression. However, Klein et al(76) suggest that the failure to induce
PC differentiation is independent of BLIMP1 expression (which they detect at similar
levels in IRF4 deficient B cells and wild type B cells), and instead attribute the
impairment to a loss in XBP1 expression.
The ability of IRF4 to initiate two distinct cell fate transitions, GC B cell and PC
differentiation, stems from its differing expression levels at these times. IRF4 is
expressed at low levels in naïve B cells but is upregulated during PC differentiation
(81). It is thought that the strength of the BCR signaling, as determined by the affinity of
the BCR for antigen, determines the level of IRF4 induction. This, in turn, determines
whether the GC B cell program or the PC differentiation program is initiated: Initial, low
concentrations of IRF4 activate AID and BCL6 expression. As the GC reaction
continues, Ig affinity increases, leading to increased BCR signaling and elevated IRF4
expression,
favoring
BLIMP1
expression(79,
82),
BCL6
repression(83)
and
extinguishment of the GC program. These divergent functions of IRF4 are mediated
through its ability to associate with different binding motifs. At lower concentrations
IRF4 cooperates with PU.1 and BATF, facilitating binding to ETS-IRF or AP-1-IRF
composite motifs and coordinating the GC program. At high concentrations, IRF4
favors binding to interferon sequence response elements (ISREs), shifting the cells
expression profile towards the PC program (79).
IRF8
IRF8 is another member of the IRF transcription factor superfamily, but unlike IRF4, is
abundantly expressed in centroblasts (84) and downregulated in centrocytes (85). IRF8
was initially proposed to positively regulate BCL6 and AID; IRF8 overexpression in
human B cells increased the abundance of BCL6 and AID transcripts, while siRNAmediated knockdown of IRF8 in a murine GC-derived B cell line had the opposite effect
(84). However, more recently, IRF8-deficient mice have been shown to display only
minor reductions in AID and BCL6 expression and have a normal antibody response
(86). Whilst the phenotype of IRF8-deficient B cells is relatively minor, knockout of both
IRF8 and its common binding partner PU.1, result in heightened PC differentiation and
class switch recombination (81). This mouse model showed that IRF8:PU.1 are
together able to help maintain the B cell program by promoting expression of PAX5
and BCL6 and concurrently repressing BLIMP1.
BACH2
BACH2 is a basic leucine zipper transcription factor, which is abundantly expressed in
both developing and mature B cells but repressed in PCs. BACH2 expression is
mediated, at least in part, by PAX5 (87, 88) and is further augmented in the GC by
BCL6 (89). One of the main functions of BACH2 appears to be the repression of
BLIMP1, reinforcing the PAX5/BCL6 mediated block on PC differentiation. As such,
BACH2-deficient B cells show inappropriate BLIMP1 expression and subsequent
impairment of AID induction and therefore CSR (90). The inhibition of BLIMP1 is
mediated through BACH2’s interaction with the protein MAFK, facilitating binding of
BACH-2:MAFK to MAFK recognition elements in the BLIMP1 promoter, suppressing its
expression(53, 91). It is likely that high levels of IRF4, resulting from strong BCR
signaling, are required to outcompete the repressive effects of BACH2 expression and
induce BLIMP1 expression(92).
FRA1
FRA1 is an AP1 family member recently identified as playing an essential role in
suppressing premature PC differentiation. Fra1 is strongly induced following B cell
stimulation and Fra1-overexpressing B cells display reduced proliferation, increased
apoptosis and disrupted BLIMP1 upregulation. FRA1 has been shown to directly bind
the
BLIMP1
promoter
and
repress
BLIMP1
induction.
Accordingly,
FRA1-
overexpressing B cells show impaired antibody production in vivo, while FRA1-deficient
B cells secrete higher titres of antigen-specific antibodies (93).
ZBTB20
ZBTB20 is a factor that has recently emerged as being required for PC longevity
following immunisation with alum adjuvant (19, 94). ZBTB20 expression, which is
dependent on high IRF4, peaks in PCs and its overexpression in B cells accelerates
PC differentiation in vitro through augmentation of BLIMP1, IRF4 and XBP1 and
reduction of BCL6. In vivo, although ZBTB20-deficient mice show no obvious
impairment in PC induction a progressive reduction in antigen-specific antibody titers is
seen, suggesting impairment in the maintenance of long-lived antigen-specific PCs
(19). Interestingly, the ZBTB20-dependent survival-defect is overridden when an
immunogen is delivered in conjunction with TLR-activating adjuvants. This suggests
that different adjuvants can activate alternate survival programs in long-lived PCs and
has implications for vaccination strategies (94).
Coordination of GC expression programs
Over the last 10 years it has become increasingly apparent that the different B cell
expression programs, activated as the GC reaction proceeds, are controlled by a highly
coordinated regulatory network. Within this network, multiple points of positive and
negative feedback ensure the mutually antagonistic expression of the “master
regulators”, augmented by an ever-increasing number of “secondary” factors that
reinforce these networks and contribute towards “sensing” the progress of the GC
reaction (Figure 3). Initially, the B cell specific expression pattern is established by
PAX5, which not only regulates the expression of proteins critical to B cell function but
also drives the expression of IRF4 (at a low level), IRF8 and BACH2. Together, these
factors inhibit the expression of the master regulators of PC differentiation, BLIMP1
and XBP1: PAX5 directly represses XBP1, while IRF8, in combination with PU.1 both
maintains PAX5 and inhibits BLIMP1. BLIMP1 is also actively suppressed by BACH2
and FRA1. Following activation of the B cell via BCR engagement, BCL6 is activated
by IRF4/PU.1. BCL6 not only controls the establishment of the GC fate, initiating the
diversification pathways and rapid proliferation of the B cells, but also further represses
BLIMP1.
Although much has been elucidated as to how these pathways repress B cell
differentiation into PCs, it is less clear how the “switch is flipped” towards favoring
terminal differentiation to PCs, essential for the final success of the GC reaction. As
SHM produces Igs of ever increasing affinity, BCR signal strength increases, in turn
increasing IRF4 expression. Increased IRF4 expression then starts to activate BLIMP1,
which in turn represses BCL6 and PAX5. This switch is further reinforced by the
activation of ZBTB20, which also enhances BLIMP1, IRF4 and XBP1 expression. Once
BLIMP1 accumulates, it represses multiple genes responsible for maintaining B cell
identity, as well as PAX5 and BCL6. This in turn allows the expression of genes
responsible for PC identity, driven in part by IRF4 and ZBTB20. Finally, suppression of
PAX5 relieves repression of XBP1, allowing establishment of the full secretory
program.
Although critical, the circuitry described above appears not to be the whole story. The
rapid proliferation of B cells is a necessary part of the GC response; but it now seems
likely that this process also plays an active role in determining cell fate. It has been
known for many years that a cell’s potential to undergo CSR is determined (at least in
part) by the number of divisions it has undergone (95, 96). Shortly after these findings,
it was shown that a B cell’s potential to undergo differentiation into an antibody
secreting cell was also dependent upon division number(97). Using ground breaking
imaging and tracking of single cell activities it has further been shown that fundamental
aspects of a B cells life (apoptosis, cell division, CSR and differentiation) are all
intimately linked to the number of times the cell has divided(98). Together, these data
suggest that B cells (and possibly all cells) posses some form of “division counting
mechanism”, that changes the cells potential to undertake the processes of cell
division, apoptosis and differentiation (reviewed extensively in Nutt et al., (38)).
Although the molecular mechanism underlying this phenomenon is not known, it likely
plays a major role in regulating the GC activities.
Much is now known about the molecular circuitry regulating the GC response and PC
differentiation, both of which are largely controlled by the expression of a small number
of “master regulators”. However, as yet no deterministic transcription factor for memory
B cells has been found. Given that longevity is a key feature of the memory response,
an alternative view is that memory B cells differentiate stochastically from GC B cells
and that a survival advantage is sufficient for memory B cell differentiation (99,
100)(reviewed in Kurosaki et al., (39)).
Conclusions
The production of high affinity antibodies of specific isotypes by PCs and generation of
long-lived memory B cells is critical to human health, not only providing protection
against the initial challenge but also shaping the immune system to generate a more
specific response upon reexposure. Conversely, the dysregulation of these processes
underlie many diseases such as autoimmune disease, allergy, lymphoma and immune
dysfunction in aging. Knowledge of these processes have allowed the development of
monoclonal antibodies that are used as diagnostic tools and to treat many diseases
and will continue towards the development of ever more successful immunotherapies.
Although much has been discovered regarding the coordination of the GC response, a
number of fundamental questions remain unanswered: What drives the re-circulation of
B cells from the light zone back into the dark zone, prolonging the GC response; how is
CSR regulated to determine the final isotype of antibody produced, and what cues
drive the cells towards terminal differentiation into memory B cells and PCs? The
unraveling of these mechanisms will no doubt provide valuable insights into the
development
of
novel
vaccine
strategies for
infectious disease
and novel
immunotherapy strategies to treat disease.
Acknowledgments
The authors acknowledge financial support from the Department of Health via the
National Institute for Health Research (NIHR) comprehensive Biomedical Research
Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's
College London and King’s College Hospital NHS Foundation Trust. TR is supported
by a studentship awarded as part of the Medical Research Council & Asthma U.K
Centre in Allergic Mechanisms of Asthma.
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Figure legends
Figure 1. Germinal center structure, formation and B cell movement.
Panel A shows the anatomical structure of the lymph node prior to commencement of
an immune response. In addition, the location of the resident cell populations and
movement of cells/immunogens are indicated. Naïve T cells (light yellow) move into the
T cell zone (paracortex) from the circulation, where resident DCs (light green) collect
and display antigen (purple triangle). Circulating naïve B cells move into B cell follicles,
located within the interfollicular region (cortex), where they sample antigen displayed
by FDCs (dark green). The location of sub-capsular macrophages (purple) is also
shown.
Panel B shows the cell interactions and movements associated with the early stages of
the immune response. Initially (day 1 of the response) naïve T cells are primed (dark
orange cell) following recognition of their cognate antigens, presented by DCs in the T
cell cortex (light pink). The T cells then move to the interfollicular regions (dark pink),
where they mature into TFH cells. Similarly naïve B cells are activated (dark blue cell)
by their cognate antigen displayed on the surface of FDCs and move out of the follicle
into the interfollicular region. By day 2 of the response antigen-primed B cells find their
cognate TFH cells to form stable interactions and become fully activated.
Panel C shows the early stages of GC formation. Cognate B/T cells move back into the
B cell follicle where B cells acquire a GC fate, undergoing rounds of rapid proliferation.
This B cell clonal expansion starts to form the GC. By day 4-5 some activated B cells
move into the sub-capsular sinus (SCS – light green) and differentiate into short-lived
plasmablasts.
Panel D. The final architecture, cellular composition and cell movement within the
mature GC is shown with the dark zone composed of rapidly proliferating centroblasts
and light zone containing centrocytess undergoing affinity maturation by selection with
TFH and FDCs. Here, non-selected cells undergo apoptosis, while some cells move out
of the GC to differentiate into long-lived PCs.
Figure 2. Regulators of GC activities and B cell fate.
Figure 2 shows the expression of the critical transcription factors that control B cell fate
at different stages of B cell maturity, from naïve B cells (light blue), through activated
GC B cells (dark blue) to plasmablasts and plasma cells. At the transition between
each stage (indicated by thick arrows) the critical change in transcription factor
expression is shown (up/downregulation of factors is indicated by small arrows). In
addition the receptors responsible for B cell movement/localisation are also shown.
Figure 3. Regulatory network controlling the GC response.
The regulatory network that coordinates the GC response is illustrated at the three
main stages of B cell differentiation, from naïve B cell, through to activated GC B cell
and finally mature plasma cell. The “master regulators” expressed in each cell type are
shown in blue boxes whilst their critical target genes/pathways is given below. The
“secondary factors” that augment the master regulators are shown above. The
regulatory interactions that exist between each of the transcription factors are depicted
by either arrows (stimulatory) or flat-headed arrows (inhibitory). Each transcription
factor and its corresponding interactions is colour coded. The activation of XBP1
brought about by the relief of PAX5 repression is represented by a dashed line.