Download 13-14 antigen specific B cell response

6. Antigen specific B cell response
Activation of both T and B lymphocytes has dual goals. Firstly, these lymphocytes protect the
body in a current infection, on the other hand these cells generate long-lived antigen specific cells
to ensure memory capacity of adaptive immunity, thus prepare the immune system for a
reinfection. While the sooner response is better in the first point, there is no time limit to create
responding cells for memory response. The goal of the secondary response, is the production of
antibodies with the highest affinity.
Accordingly B cell activation results in:
1. early production of antibodies, to recognize and neutralise the antigen or to opsonize it to
facilitate the innate immune reactions.
2. the production of memory B cells and long-lived plasma cells, which cell populations
ensure the high affinity interaction with the antigen.
1. Antigen entry to the secondary lymphatic organs
Naive lymphocytes practically encounter the antigens in the secondary lymphoid organs, which
provide the proper environment for the extreme proliferation of both types of lymphocytes. For B
cells, mostly follicular dendritic cells (FDCs) display the antigens in the germinal centers, but it is
not yet fully clear how do the antigens enter the follicles and how are positioned on the FDCs.
Unlike T cells, B cells are able to recognize nearly all molecule types, ranging from living bacteria
to degraded products of macromolecules. The endothelium lining is not tightly packed in the
sinusoid layer of the lymphatic vessels in the lymph nodes, thus smaller antigens can move
directly to the secondary lymphatic organs. Larger antigens can get to the lymph nodes as part of
the immune complexes. The complexes seem to be predominantly cell surface associated.
Antigens opsonized by immunoglobulins or complement proteins are fixed to the cell surface by
Fc and complement receptors. Macrophages, dendritic cells, non-antigen specific B cells, capture
immune complexes in this way and deliver the antigen to the lymph nodes. (Red blood cells play
also crucial role in the transport of blood borne immune complexes to the spleen) Antigens
arriving at the lymph node localize to the surface of FDCs, where they will be arrested due to high
complement and Fc receptor expression on FDCs. Once immune complexes reside on FDCs,
FCDs have the unique property of retaining them on their surface for long periods of time. In
summary the transport of immune complexes and their attachment to the FDCs are regulated by
non-antigen specific receptors.
2. T-independent B cell response
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B cells are able to recognize various antigen substances, T cell activation is restricted to peptide
recognition (presented by MHC molecules). Accordingly, T-dependent and T-independent B cell
responses are highly different. Effective B cell memory response cannot be generated without the
help of T cells, thus, memory B cells develop principally to protein antigens.
B cell response will be less intense in the absence of T cell-mediated helper signals, than the Tdependent response. This process results in low affinity antibodies, defective in class switching
and generates weak memory response. Two main classes of T-independent responses are
distinguished:
T-independent I (TI-1): PRRs or complement receptors expressed on the surface of B lymphocytes
can recognize the antigen or opsonins attached to it. These co-receptors signalise simultaneously
with the BCR in a synergistic manner.
T independent II (TI-2): B-cell response to TI-2 antigens are critically dependent on the formation
of high number of antigen receptor clusters, generating extremely intense BCR signal.
B1 B cells and marginal zone B cells are the main types of the T-independent response:
B1 cells are a unique B cell population with innate like function. Unlike other B cells, B1 cells
produce antibody independently of the presence of antigen. These natural antibodies recognize
conserved, frequently expressed molecules of pathogens, similarly to the function of PRRs. As a
result of these processes, these natural antibody molecules present in the serum prior to infection.
B1 cells produced antibodies are low affinity IgM molecules and recognize the same antigens in
everyone, being specific to conserved motifs.
Marginal zone B cells localize characteristically in the marginal zone of the spleen. These B cells
are activated firstly upon the appearance of the antigen; they differentiate to plasma cells and
produce antibodies in 1-2 days. In this case, the produced antibodies are also low affinity IgM
molecules (IgG can appear).
3. T-dependent B cell activation
Due to the interaction with T cells, a part of B cells differentiate into extrafollicular plasma cells to
provide quick protection, other part of B cells differentiate to memory B cells or long-lived plasma
cells in a multi- step, time consuming process in germinal centers.
3.1 Extrafollicular B cell activation
Hematopoiesis results in a B-cell repertoire of around 107-9 distinct binding specificities daily in
the bone marrow, directed by the mechanism of V(D)J recombination. This mechanism occurs
independently of antigens and results in the generation of clonally defined variable region in both
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the heavy and light chain Ig genes. The primary repertoire has such complexity, that only a very
small fraction (less than 0.01%) of the B cell clones are recruited into a response against particular
foreign epitopes.
T-dependent B cell activation may result in B cell activation in extrafollicular focuses or in GCs
within the B cell follicles. The highest affinity B cells differentiate to extrafollicular B cells upon
antigen recognition, while the lower affinity, but still antigen specific B cells enter the germinal
centers.
B cell proliferation begins within 1–2 days at the T zone–follicle junction of the germinal centers,
where the activated B cells are distributed in a perifollicular pattern. Upon antigen recognition
extrafollicular B cells proliferate, and differentiate into plasma cells in a few days. The key role of
the extrafollicular activation is the early activation of B cells, this early antibody response
facilitates the pathogen clearance and creates immune complexes. Extrafollicular B cell activation
can bring about the differentiation into memory B cells, and isotype switching from IgM to IgG,
during the perifollicular B cell proliferation phase, but these early B cells have not yet undergone
SHM, thus do not improve the affinity of antibodies during the response.
Extrafollicular B cell activation is a type of T dependent B cell response, to protect the body in a
current infection. Following the encounter with an arriving antigen, the activated B cells move
toward the T cell zone and present the specific antigen to T cells, which have already been
activated by DCs. B–T cell interactions are not limited to antigen-specific (cognate) interactions
between the T cell receptor (TCR) and peptide-MHCII on B cells. The TCR-induced membrane
protein CD40 ligand (CD40L), that activates CD40 receptor on B cells, is the most important T
cell–derived signal. CD40L signal promotes the survival and the proliferation of B cells, and it is
required for isotype switch and somatic hypermutation. In addition to cell–cell interactions, CD4+
T cells provide help for B cell activation by secreting cytokines, including IL-4 and IL-21.
3.2 The development of secondary follicles
While the originally high-affinity B cells provide rapid protection, the low-affinity, but antigen
specific B cells accumulate as germinal center cells. This part of antigen specific B cells enters to
the primary follicles upon the interaction with T cells and following clonal proliferation establish
secondary follicles, germinal centers (GC). GC are normally transient structures. GC B cells
appear rapidly, within 48 h in some responses, but the follicles typically reach maximum size 1–2
weeks after the delivery of an antigen. Even though a large number of naive B cells out have the
cell-intrinsic potential to go into a GC reaction (polyclonal response), entry into the GC is a
competitive procedure. The presence of high-affinity competitors inhibiting the activation of
lower-affinity B cells prior to GC formation. Higher-affinity cells arriving to the GCs can
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efficiently outcompete the original clones, ultimately replacing the lower-affinity clones that
initiated the reaction. Finally, GCs are colonized by a very small number of high-affinity
precursors. (oligoclonal response).
3.3 The role of FDC in antigen specific B cell reactions
B cells are selected by antigens displayed on the FDC surface in the form of immune complexes.
FDCs are characteristic stromal cells within lymph nodes. FDCs are just named dendritic cells, due
to similar morphology, but they have completely different function. These cells are not derived
from myeloid progenitors in the bone marrow and they do not express MHC class II molecules and
only a very few of PRRs. FDCs practically cannot recognize free antigens, but are very good at
binding and retaining opsonized antigens.
FDCs have only a minor role in B cell homeostasis within resting lymph nodes, forming reticular
networks in primary follicles. However, FDCs play a fundamental role in secondary follicles upon
antigen exposure:

FDCs act as long-term reservoirs of intact antigens,

provide survival and co-signals for the B cells and direct their movement, both by cell
surface receptors and cytokines
FDCs act as long-term reservoirs retaining intact antigens on their surface in the form of
immune complexes (so called iccosomes). Immune complex trapping relies primarily on
complement receptors and secondly by Fc receptors. FDCs can retain antigens and display it to B
cells for extended periods of time, even for several months.
Immune complexes on FDCs bring the possibility that GC B cells receive multiple stimulatory
signals that depend on their interaction with the antigen, but that are delivered via receptors other
than the BCRs. These include signals delivered from complement fragments deposited on the
immune complexes via complement receptors on B cells. These signals are likely to make an
important contribution to the selection of high-affinity B cells in the GC. In addition, synapses are
formed between FDCs and B cells, which are ensured by the interactions of complementary pairs
of intercellular adhesion molecules such as ICAM-1 ⁄ LFA-1 and VCAM-1 ⁄ VLA-1.
FDCs are thought to support GCs by secreting chemokines and cytokines that attract and sustain
GC B cells. CXCL13 is the major chemoattractant both for B cells and special T cell
subpopulation, the follicular helper T cells (TFH). This chemokine helps for CXCR5 expressing B
cells to localize in the GC. FDCs produce an array of cytokines, most notably IL-6 and BAFF,
both of which may play a role in the GC reaction, supporting B cell survival and coordinating the
germinal center reaction.
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3.4 Follicular helper T cells
GCs predominantly consist of B cells but also contain FDCs and CD4+ T cells. (Specialized form
of macrophages can also be found in the GC, these cells able to engulf and eliminate apoptotic B
cells, those are frequent products of GC selection.)
Only a small fraction of the cells in GCs are T cells (Approximately 5–20%), nevertheless, these
cells are essential for GC maintenance and for affinity maturation. As for the extrafollicular
interactions with T cells at the initiation of B cell activation, the interplay with T cells is also
required for GC formation and for selection of somatically mutated GC B cells. The specific T cell
population in the GC is known as T follicular helper (TFH) cells. Activation of CD4+ T cells allow
these T cells to enter into the B cell follicle. TFH cells express special costimulatory receptors to
regulate the interaction with B cells. (For example inducible T cell co-stimulator (ICOS),
programmed cell death 1 (PD1) and signalling lymphocyte-activation molecule (SLAM)) These
interactions with the appropriate receptors on B cells promote the differentiation of T cells into
TFH cells and play a crucial role in IL-21 production. The cell-cell interactions between B cells and
helper T cells together with IL-21, produced by TFH cells, are essential for survival and
proliferation of GC B cells, and direct plasma cell differentiation of B cells. TFH cells also release
other kind of cytokines to determine the isotype of the antibodies during class switch.
3.5 B cell selection in germinal centers
The time consuming selection of the highest affinity B cells takes place in the germinal centres of
the secondary lymphatic tissues. B selection in GCs operates on the basis of the relative affinity of
competing clones. GC B cells with the highest affinity for an antigen, preferentially receive
survival and proliferative signals, guiding their selection. Low-affinity cells are incapable of
forming GCs in the presence of higher-affinity competitors, since high affinity cells can consume
all antigens or at least block access to the antigen rich sites on FDCs. The amount of antigen
available for lower affinity cells would be insufficient to trigger a BCR signal strong enough to
rescue these cells from apoptosis, and the absence of BCR-mediated signaling leads to their
elimination.
BCR stimulus alone is not sufficient for affirmative selection, without T cell help it rapidly kills
rather than propagates GC B cells. Proliferation of antigen-activated B cells requires also CD4+ T
helper-mediated signals. T cell provides activator signals only for those B cells, which present
antigen to the antigen specific T cells. Antigen presentation to the T cells requires BCR-mediated
internalization of foreign antigen, its processing into peptide fragments, and its presentation to
cognate T cells residing in the GCs conjunction. The BCR captures and mediates the
internalization of different amounts of antigens from FDCs, in proportion to its affinity. BCR
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affinity determines not only the intensity of BCR signaling, but also the effectivity of antigen
presentation. T-cell signals are primarily delivered via CD40 ligand as well as T cell derived
cytokines, such as IL-4, IL-5, IL-13, and IL-21.
In summary higher affinity B cells
(1) can encounter more antigens on the surface of FDCs, competing with other opponent,
(2) receive stronger BCR signals,
(3) present more antigens to Th cells and thus receive more Th signals.
Only B cells that obtain both BCR signals and T cell help can survive, and start proliferation. The
reduced ability of low-affinity B cells to compete with high affinity B cells, could be due to
impaired access to antigens, or T cell help, or both of these signals.
3.6 Somatic hypermutation and affinity maturation
Antigen-induced BCR signalization and the simultaneous T cell-mediated helper signals results in
the selection of high-affinity B cells. The highest affinity B cells are the winners of the
competitions for antigens, and are able to present the antigen for T cells effectively. Interactions
with T cell activate the CD40-induced signaling, which leads to the synthesis of activation-induced
deaminase (AID) in B cells, an enzyme that is required for somatic mutation and isotype
switching.
Somatic hyper mutation (SHM) leads to diversification of Ig variable region. Beside the bone
marrow localized mechanism of V(D)J recombination, SHM also has an enormous impact on
shaping the BCR repertoire. Point mutations in antibody V regions are induced due to AID activity
in B cells, as a result, yield different affinities to the developing clones. (In plasmablasts, Ig V
genes undergo point mutations at an extremely high rate, which are about a million times higher
than the spontaneous rate of mutation in other mammalian genes.) SHM is a random process
generating profitable but also unprofitable mutations. After clonal proliferation and somatic
mutations, the generated clones compete for antigen access and for T cell help again. Those, that
have improved affinity to the antigen during SHM compete over the strength lost clones and
dominate antibody production. Whereas higher-affinity cells are selectively expanded, loweraffinity cells are eliminated by apoptosis. In GCs B cell clones have an increasingly higher average
affinity for the immunizing antigen, calling the process ‘affinity maturation’. At the end of the
process the affinity of GC B cells go high above the affinity of extrafollicular B cells.
3.7 Isotype switch
Interaction with T cells also induces isotype switch, a process that modulates the effector functions
of the produced antibodies. Firstly, naïve B cells express immunoglobulins IgM and IgD classes,
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but after activation they can change their heavy chain classes to IgG, IgE, and IgA. The rearranged
(VDJ) variable region is not changed, thus class switching does not affect antigen specificity. AID
expression and the initiation of class switch are regulated mainly by CD40 signals. (due to the
interaction with CD40L expressed on T cells) In the meantime the direct regulation of which CH
region will be transcribed is determined by the cytokine environment. For example the IL-4
production of helper T cells leads to switch to IgE, IFNγ to IgG and TGFβ to IgA.
3.8 Cyclic movement of B cells between dark zone and light zone
The GC is functionally polarized into

a dark zone (DZ) in which B cells divide and

a light zone (LZ) in which B cells are activated and selected based on their affinity to the
antigen.
The DZ consists almost entirely of B cells with a high nucleus-to-cytoplasm ratio, thus appearing
“dark” by light microscopy. B cells in the DZ were referred to as “centroblasts” are among the
fastest dividing mammalian cells, with a cell-cycle time estimated between 6 to 12 h. In contrast, B
cells in the LZ are mingled among T cells and the network of FDCs. As antigen is deposited on the
FDC network, the presence of antigens is characteristic only in the LZ. The regulation of B cell
activation and selective T cell signals for high-affinity mutants located in the LZ of the GC
Antigen engagement and selection are executed in the LZ while cell proliferation and SHM passes
in the DZ, which requires that GC B cells transit between the two zones. Accordingly, B cells were
found to constantly migrate between the two GC compartments. A fraction of B cells positively
selected by survival and activation signal in the LZ would return to the DZ for further rounds of
proliferation and mutation, and go again to the LZ for selection in a cyclic fashion. The decision to
return to the DZ and undergo clonal expansion is controlled by T helper cells in the LZ, which
discern between LZ B cells based on the amount of antigen captured and presented. B cell affinity
increases in a stepwise fashion with each additional cycle, suggesting multiple rounds of selection.
3.9 Generation of long-lived plasma cells and memory B cells
The positively selected germinal center B cells have three options following the interaction with T
cells. These cells may start a (1) new cycle re-entering the GC reaction for further diversification,
finally GC cells can be exported from the GC as (2) plasma cells or as (3) memory B cells. These
two cell types ensure protection almost exclusively in reinfection.

Long-lived plasma cells
As a final outcome, GC B cells can differentiate into memory B cells or plasma cells. Long-lived
plasma cells persist and provide protection in the absence of antigen re-exposure for years.
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The long-lived plasma cells that reside in the bone marrow are known to have heavily somatically
mutated variable region genes and thus produce high-affinity antibodies, suggesting that affinitybased selection does play a prominent role in their production. Highest affinity GC B cells are
preferentially selected to undergo plasma cell differentiation. Enhanced BCR signaling is one
candidate, as this has been shown to trigger plasma cell differentiation. However, the enhanced
provision of T-cell help to GC B cells also drives a burst of plasma cell production. In addition to
CD40, other T cell–derived signals can affect plasma cell differentiation in vivo such as IL-21mediated signals. The differentiated plasma cells do not express BCR, thus are unable to sense
antigens, but release antibody molecules constantly. The released high affinity antibody molecules
provide immediate protection upon reinfection, resulting in neutralization and opsonization of
antigens.
Most of the GC-derived plasma cells migrate to the bone marrow or local mucosa-associated
lymphoid tissues. The bone marrow niche supports plasma cell survival through cooperative
survival signals from stroma-derived cytokines.

Memory B cells
are activated following reappearance of antigen. Contrary to plasma cells these cells express
antigen receptor and only following antigen recognition start to proliferate and differentiate to
plasma cells or create the next generation of memory cells.
Memory cells can be generated from T cell-B cell interactions in a GC-independent manner,
nonetheless, the majority of memory cells in wild-type mice responding to T-dependent antigens
are likely to arise from the GC reaction. The presence of SHM in Ig genes is characteristic in these
memory B cells, although memory cells are of lower affinity than plasma cells. Post-GC memory
B cells are not as specific as high affinity plasma cells, but overall, affinity of the memory B cells
increases in line with the GC response. As opposed to terminally differentiated plasma cells, it
may be advantageous to maintain relatively low-affinity memory B cells to provide the flexibility
to respond to variant pathogens that have mutated the initial target antigen.
4. Autoimmune reaction
B cells that acquire reactivity with self-antigens are an undesirable but an inevitable product of the
GC response and may be the basis of an autoantibody response. Because Ig SHM is a random
process there is a continual risk to create or increase antibody affinity for self-antigens. The
production of dangerous autoantibodies in the GC must be tightly controlled. Isolated cross-linking
of the BCRs on GC B cells leads to rapid cell death in the absence of either FDC-associated cosignals or TFH help. GC B cells that preferentially interact with self-antigens in the GC will also be
unable to access the co-signals associated with FDCs or TFH help. B cells that bind self-antigens
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would not receive signal due to the absence of self-reactive T-cell help and due to the absence of
self-antigens on the FCDs. Most of the autoreactive B cells either created during SHM or entering
the GC from the circulation die by apoptosis in the absence of the aforementioned survival signals.
There is the possibility however, that GC B cells undergo somatic mutations that increase their
affinity for foreign antigen and in the meantime acquired them self-reactive BCRs , creating crossreactive specificities. Cross-reaction leads to the development of autoimmune B cells, especially
when expression of the target self-antigen is either extremely low or absent from the GC, such as
for a tissue-specific protein. In this case, recognition of FDC-associated foreign antigen and
signals from foreign antigen specific TFH cells would also support these B cells resulting in their
differentiation into plasma cells and the production of high-affinity antibodies directed against the
original foreign antigen. However, once released into extracellular fluids, these same antibodies
would also be free to access and bind the distal self-antigen targets, potentially contributing to an
organ-specific autoimmune disease. Accordingly, high-affinity heavily mutated antibodies have
been found in patients suffering different autoimmune diseases.
In summary, even though the germinal center reactions are highly controlled by the balance of
survival and apoptotic signals for B cells, somatic mutations highly increase the risk of
autoimmune reactions.
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