Download maturation of humoral immune responses

Doctorial Thesis from the Department of Immunology
The Wenner-Gren Institute, Stockholm University
Stockholm, Sweden
MATURATION OF HUMORAL IMMUNE RESPONSES
STUDIES ON THE EFFECTS OF ANTIGEN TYPE,
APOPTOSIS AND AGE
KARIN LINDROTH
STOCKHOLM 2004
Summary
The humoral immune response is dependent on the formation of antibodies. Antibodies
are produced by terminally differentiated B cells, plasma cells. Plasma cells are
generated either directly from antigen challenged B cells, memory cells or from cells
that have undergone the germinal center (GC) reaction. The GC is the main site for class
switch, somatic hypermutation and generation of memory cells.
Different factors, both internal and external, shape the outcome of the immune
response. In this thesis, we have studied a few factors that influence the maturation of
the humoral response. We have studied how age affects the response, and we show that
responses against thymus dependent antigens (TD) are more affected than responses to
thymus independent (TI) antigens, in concordance with the view that the T cell
compartment is more affected by age than the B cell compartment. Furthermore, we
demonstrate that priming early in life have a big influence on the immune response in
the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx)
induces a reduction of IgG levels in later TD responses against Dx. We have evaluated
possible mechanisms for this reduction. The reduction does not seem to be caused by
clonal exhaustion or antibody mediated mechanisms. We also showed that the reduced
TD response after TI priming can be induced against another molecule than Dx. With
the hypothesis that TI antigens induce a plasma cell biased maturation of the responding
B cells, we examined the presence of Blimp-1, a master regulator of plasma cell
differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs
induced by TI antigen and the staining intensity in these GCs was stronger than in TD
antigen induced GCs, indicating that plasma cells might be continuously recruited from
these GCs.
B cells undergoing the GC reaction are thought to be under a strict selection
pressure that removes cells with low affinity for the antigen and also cells that have
acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the
accumulation of somatic mutations in GC B cells. In mice lacking the death receptor
Fas, lpr mice, the frequency of mutations was increased but the pattern of the mutations
did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic
protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had
other characteristics.
© Karin Lindroth
ISBN 91-7265-835-5
Akademitryck AB, Edsbruk 2004
Stockholm 2004
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TABLE OF CONTENTS
ORIGINAL PAPERS
Abbrevations
INTRODUCTION TO THE IMMUNE SYSTEM
Innate and adaptive immunity
Lymphoid organs and tissues
Hematopoiesis
T cells
B CELL IMMUNOLOGY
Development to mature B cell
Immunoglobulin - the multifunctional receptor of the B cells
IgM
IgD
IgG
IgA
IgE
Fc receptors
Activation signals
Co-receptors
Complement receptor 2
FcγRIIB
B cell responses against TD antigens
B cell responses against TI antigens
Affinity maturation
Class switch
The germinal center reaction
Plasma cell differentiation
Death and survival of mature B cells
Humoral immunity
Humoral responses and aging
PRESENT STUDY
AIM
METHODOLOGY
Mice
Immunizations, Antigens and Adjuvants
ELISA
Estimating antibody affinity
Immunohistochemistry
Generation of hybridoma cell lines
Extraction of GC B cells from Peyer’s patches
DNA and RNA isolation, RT-PCR amplification and sequencing
RESULTS
Paper I
Paper II
Paper III
Paper IV
CONCLUDING DISCUSSION
ACKNOWLEDGEMENTS
APPENDIX: PAPER I-IV
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Original papers
This thesis is based on the following papers, which will be referred to in the text by their
Roman numerals:
I
Marta Sánchez, Karin Lindroth, Eva Sverremark, África González Fernández,
Carmen Fernández. The response in old mice: positive and negative immune
memory after priming in early age. Int. Immunol. 2001.13:1213-1221
II
Karin Lindroth, Elena Fernández Mastache, Izaura Roos, África González
Fernández and Carmen Fernández. Understanding thymus independent antigen
induced reduction of thymus dependent immune responses. Submitted
III
Karin Lindroth and Carmen Fernández. The role of Blimp-1 in the GC
reaction. Differential expression of Blimp-1 upon immunization with TD and TI
antigens. Submitted
IV
Elena Fernández Mastache*, Karin Lindroth*, Africa González-Fernández¤ and
Carmen Fernández¤. Somatic hypermutation of Ig genes is affected differently
by failures in apoptosis caused by disruption of Fas (lpr mutation) or by overexpression of Bcl-2. Submitted
*
E.F.M. and K.L share first authorship. ¤ A.G.F. and C.F share senior authorship
-5-
Abbreviations
AID
APRIL
BAFF
BAFF-R
Bcl
BCMA
BCR
Blimp-1
BLNK
BM
BSA
BSAP
C
CDR
CR
CT
D
DNP
Dx
EBF
Fas
FDC
FR
GC
HSC
Ig
IL
IRF4
ITAM
ITIM
J
lpr
LPS
MHC
NK
Ox
PALS
PI3K
PLCγ2
PNA
SHM
TACI
TCR
TD
TH
TI
TI-1/TI-2
TLR
V
XBP-1
XID
activation-induced deaminase
a proliferation inducing ligand
B cell activating factor
BAFF receptor
B cell lymphoma
B cell maturation antigen
B cell receptor
B lymphocyte-induced maturation protein I
B cell linker protein
bone marrow
bovine serum albumin
B cell lineage-specific activator
constant
complementarity determining region
complement receptor
cholera toxin
diversity
dinitrophenyl
dextran B512
early B cell factor
FS-7 associated surface antigen
follicular dendritic cell
framework region
germinal center
hematopoetic stem cell
immunoglobulin
interleukin
interferon-regulatory factor 4
immunoreceptor tyrosin-based activation motif
immunoreceptor tyrosin-based inhibitory motif
joining
lymphoproliferation
lipopolysaccharide
major histocompatibility
natural killer
oxazolone
periarteriolar lymphoid sheath
phophatidylinositol 3-kinase
phospholipase Cγ2
peanut agglutinin
somatic hypermutation
transmembrane activator and CAML-interactor
T cell receptor
thymus-dependent
T helper cell
thymus-independent
thymus-independent type 1/ thymus-independent type 2
Toll-like receptor
variable
X box-binding protein 1
X-linked immunodeficiency
-6-
Introduction to the immune system
An organism is constantly threatened by external and internal challenges. These threats
have acted as evolutionary pressures leading to the emergence of various protective
mechanisms. In vertebrates, mechanical barriers like skin and mucosa act as a first way
to stop the entry of a wide variety of harmful organisms, pathogens, e.g. bacteria,
viruses and parasites. The body also possesses physiological barriers to further
complicate for the invading organism, e.g. the body temperature and the pH of the skin
and body fluids. Furthermore, if a pathogen passes these barriers, it will encounter a
diverse set of cells and molecules that have the capacity to recognize and eliminate the
invading organism. Some of these defense mechanisms also have the ability to act
against internal challenges such as transformed cells, which could otherwise lead to
cancer growth. All these elements are functionally specialized parts that together make
up what we call the immune system.
Innate and adaptive immunity
The immune system is often described as consisting of two branches, one innate and
one adaptive. The innate (or natural) immune system consists of the body’s mechanical
and physiological barriers as well as effector cells and molecules. Distinctive for all
these mechanisms is that they are not specific for a particular pathogen, either working
unspecifically, i.e. the anatomical barriers, or recognizing elements that are conserved
and used in a large group of pathogens, e.g. recognition of molecules in the cell wall of
bacteria by pattern-recognition receptors. Although the innate immune system can
produce a very rapid and strong response it lacks the ability to adapt to a specific
invading pathogen. The innate immune system also lacks the ability to form an
immunological memory, which is the capability to elicit an improved response after
previous antigen exposure, a feature of the adaptive immune system.
While the recognition by the innate immune system is conserved, one hallmark of the
other branch of the immune system, the adaptive (or acquired), is its capacity to react
specifically against a pathogen. This is due to the vast ability of the adaptive immune
system to genetically create receptors with different specificities. These receptors are
produced by specialized cells called B and T lymphocytes. Fundamental for the function
of the adaptive immune response and also for the formation of memory is that one
lymphocyte only carries one type of antigen-specific receptor, and that this one
lymphocyte can proliferate and generate a number of identical cells, a clone, carrying
the same receptor. When activated by an invading pathogen, the responding
lymphocytes can exert a variety of effector functions. B lymphocytes produce a soluble
variant of their receptor, an antibody, which when secreted helps to neutralize and
eliminate the pathogen and also triggers some of the innate immune responses. T
lymphocytes can mature into cytotoxic T cells, that can actively kill infected cells, or T
helper (TH) cells which help the activation and maturation of B and T cells. After the
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infection is cleared, some of the lymphocytes with receptors specific for the antigen will
remain in the body as so called memory cells. When encountering the same antigen
again, these memory cells will make the immune response faster and of higher
specificity than the first response. Both adaptive and innate immune responses are
needed for an optimal response against pathogens, and the two systems interact with
each other.
Lymphoid organs and tissues
The immune system operates in several specialized environments. Their function is both
to provide a microenvironment that can support the maturation of clonally diverse
lymphocytes from pluripotent precursors, as well as providing the right context for
bringing lymphocytes in contact with their antigen. The produced effector and memory
cells must also be able to migrate to the appropriate sites. The lymphoid tissues are
often divided into primary, secondary and tertiary. Examples of primary lymphoid
tissues are bone marrow (BM) and thymus, which support the differentiation of
progenitors to functionally mature B and T cells respectively. Secondary lymphoid
tissues constitute the environment where naïve lymphocytes first meet exogenous
antigen, and support the activation of naïve cells and the emergence of expanded
effector and memory cell populations. This takes place in lymph nodes, Peyer’s patches
and the spleen. Tertiary lymphoid tissues are the tissues with immune effector functions,
the “battlefield”. These could be any tissue in the organism, but most often are tissues
frequently exposed to the surroundings, such as skin and mucosa.
Hematopoiesis
The effector cells of both the innate and the adaptive immune system have a common
origin. They arise from hematopoetic stem cells in the BM that have the capacity to
produce cells of all blood-cell lineages (figure 1). This is possible since the
hematopoetic stem cells have the capacity to self-regenerate as well as allowing further
differentiation of daughter cells. An early option of the differentiating cell is to develop
into a myeloid or lymphoid progenitor cell. The myeloid progenitor cell can in its turn
diverge to forming red blood cells, platelets or phagocytic cells (monocytes and
granulocytes). Phagocytic cells have the ability to engulf and digest microorganisms.
The lymphoid progenitor on the other hand can further differentiate to and form natural
killer (NK) cells, B and T lymphocytes.
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Red blood cells
Platelets
Monocytes
Myeloid
progenitor
Granulocytes
Lymphoid
progenitor
B cells
HSC
T cells
NK cells
Figure 1.
Hematopoiesis. The hematopoetic stem cell (HSC) give rise to
myeloid and lymphoid progenitors which in turn can differentiate
further to form specialized cell types.
T cells
T cells share a common progenitor in the BM with B cells; however their development
into functionally competent cells takes place in a different organ, the thymus. In the
thymic environment the T cell progenitors rearrange the genes coding for their antigen
specific receptor. The process in which the cell through rearrangement forms its antigen
receptor is closely linked to two selection processes. One process, the positive selection,
ensures that the becoming T cell only recognizes antigen in the form of processed
peptide bound to the surface of major histocompatiblility complex (MHC) class I or
class II molecules. The other process, the negative selection, eliminates those cells that
show reactivity against self molecules, thereby removing cells that could potentially
cause autoimmune reactions [reviewed in Palmer 2003]. When leaving the thymus the
majority of T cells will carry a receptor that consists of the T cell receptor (TCR) α and
β chain associated to the CD3 complex, a few cells will express the TCR γ and δ chains.
In this complex, the TCR chains are responsible for recognition of antigen (peptide+
MHC class I/II) while the intracellular signaling is conveyed by the CD3 subunits
[reviewed in Alarcon et al 2003].
The emerging T cells can further be divided into subsets based on the expression of the
CD4 or CD8 co-receptors. CD8+ T cells recognize antigen when it is processed and
presented on a cell surface by MHC class I molecules. Almost all nucleated cells
express these molecules, in contrast to MHC class II molecules that are only
-9-
constitutively expressed by a group of cells called professional antigen presenting cells,
including macrophages, B cells, dendritic cells and thymic epithelial cells. Peptides
presented by MHC class II are the recognition element for the other T cell subset, the
CD4+ T cells. Due to differences in the process of peptide presentation, MHC class I
molecules most often present peptides that have been produced by the presenting cell,
while the MHC class II molecule presents peptides that have an extra-cellular origin.
This is also reflected in the effector functions of the different T cell subsets. CD8+ T
cells have cytotoxic functions and can thus eliminate cells that present internally
produced foreign peptides on their surface, e.g. a virus infected cell. The CD4+ T cells
on the other hand are potent activators and modulators of other immune cells through
the use of different co-stimulatory molecules and soluble factors e.g. interleukins (IL).
These cells are often referred to as TH cells. Interactions between T and B cells are
important for several different phases in the humoral immune response. They will be
discussed in more detail in their biological context.
B cell immunology
Development to mature B cell
Unlike T cells, the development of B cells from progenitor stem cells to functionally
mature B cells takes place in the BM. The B cell progenitor assembles the Ig genes
during its development in the BM, and different stages of B cell development have been
characterized in part based on which stage in the rearrangement process the cell is in
[Hardy et al 1991; reviewed in Karasuyama et al 1996; reviewed in Osmond et al
1998]. The development of B cells is characterized by successive rearrangements, first
of the gene for the Ig heavy chain and then the gene for the light chain. Originally, the
loci for the variable (V) region of the Ig chain contain different elements that will be
part of the assembled gene. The heavy chain region has V, diversity (D) and joining (J)
elements, and the light chain region V and J elements. The assembly of the heavy chain
V region occurs in two steps, first is one of the DH elements joined with a JH element,
and then a VH element is joined to the combined DJ region to complete the gene (figure
2).
When B cell precursors rearrange the VH to the DHJH-rearranged alleles, the
rearrangement can be either in or out of frame. An in-frame rearrangement leads to
expression of an Ig heavy chain with the potential to form a pair with a surrogate light
chain forming a pre-B cell receptor (BCR) on the cell surface. Cells that have been able
to express a pre-BCR undergo two important events. The VH to DHJH rearrangement at
the second heavy chain allele is stopped, a process leading to allelic exclusion. This
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VH
DH
JH
Cµ
germline
VH
DH JH
Cµ
DJ rearranged
VH
DH JH
Cµ
VDJ rearranged
Figure 2.
Rearrengement of the Ig heavy chain genes. In the first step a
D and a J element are joined. To complete the gene the DJ
region is joined with a V element.
prevents the B cell from expressing two different heavy chains. Furthermore, a
proliferative expansion is induced in the cells carrying the pre-BCR, a kind of positive
selection for pre-B cells with a productive VHDHJH rearrangement. After this
proliferative burst the cells will lose the expression of the surrogate light chain and start
to rearrange the light chain genes. In this case a V region is joined directly to a J
element. This assembly of the V genes contributes immensely to the diversity of
antigen-binding specificities. The presence of multiple germline V, D, and J elements
creates a large number of different possible combinations. Furthermore, the elements
can be joined at different positions and extra nucleotides can be inserted in the
junctions, thus creating a huge potential for constructing unique receptors.
Cells that have managed to produce a light chain rearrangement, resulting in an
expressed polypeptide chain that can pair with the heavy chain, will become immature
B cells, displaying an IgM receptor on the surface. These immature B cells will undergo
a negative selection aiming to remove B cells that recognize self antigens. If an
immature B cell encounters its antigen it will result in down regulation of the expression
of IgM and B220, and an arrest of further differentiation [Hartley et al 1993].
Recognition of antigen can also result in receptor editing, when the immature B cell
further rearranges the light chain with the chance of creating a non-auto reactive
receptor and thus avoid negative selection [Gay et al 1993; Radic et al 1993; Tiegs et al
1993].
To develop into a mature B cell, the immature B cell leaves the BM and head out to
lymphoid organs in the periphery, where it has the possibility of encountering antigen
and differentiating into either a memory or a plasma cell. If not receiving the signals
necessary for maturation, the half-life for an immature B cells is measured in days. In
fact only 5-10% of the newly generated immature B cells get selected into the pool of
mature B cells [reviewed in Osmond 1993].
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Immunoglobulin - the multifunctional receptor of the B cells
The characteristic molecule of the B cells is the antigen receptor, the immunoglobulin
(Ig). The Ig exists both as a membrane bound receptor, the BCR, as well as in a secreted
form that is produced by activated B cells, plasma cells. As surface receptor the Ig is
involved in such fundamental cellular processes as differentiation, activation and
apoptosis, while the secreted form is capable of both directly neutralizing foreign
antigen as well as recruiting other effector systems.
The Ig consists of four polypeptide chains paired together, two larger chains, called
heavy chains, and two shorter chains, the light chains (figure 3). Each chain is built up
of several smaller units called immunoglobulin homology domains. The Ig light chain
contains two such Ig domains, the heavy chain has either four or five depending on the
isotype of the antibody.
Antigen binding
VL
Figure 3.
The immunoglobulin (Ig). The Ig is
formed by two light and two heavy
chains paired together. The variable
(V) parts of the Ig are the part that
bind to antigen while the constant
(C) regions carry the other functions
of the Ig molecule.
VH
CL
s
CH1
s
s
s
s
Fc part
CH2
s
CH3
Due to great sequence variability the amino-terminal part of each chain is termed a V
region, VH and VL for the heavy and light chains respectively, and is the region that
carries the antigen specificity. The antigen binding part of the antibody can be further
divided into areas with high or low sequence variability. A V region has four more
conserved motives called framework regions (FR1-4), which are separated by the highly
variable complementarity determining regions (CDR1-3). When the protein folds the
CDR regions come together and form the antigen-binding surface. The interactions
between the antibody and the antigenic surface are formed through hydrogen bonds,
electrostatic and Van der Waals interactions as well as hydrophobic forces [reviewed in
Mariuzza et al 1994]. The sequences of the other Ig domains are not so variable and are
consequently called constant (C) regions. The light chain only have one C domain,
while the heavy chain can consist of three or four C domains depending on isotype.
- 12 -
The C region of the heavy chain is responsible for the functions of the antibody apart
from antigen binding, for example interacting with receptors and complement and the
ability to multimerize. There are five different classes of heavy chain C regions, each
corresponding to an Ig isotype with characteristic effector functions. The Ig isotypes,
IgM, IgD, IgG, IgA and IgE, can appear both as receptors on the cell surface and in a
secreted form, with each C region class giving different specific functions to the Ig
molecule. During an antibody response there is often class changes of the antibodies
produced. However, although an individual B cell can be induced to produce different
Ig classes during its life time, each B cell will only produce Igs with identical binding
sites for the antigen, Igs with identical antigen specificity.
IgM
IgM is the first isotype expressed during B cell development, and is the major isotype in
primary antibody responses. In sera 5-10% of the Igs are of this isotype, and IgM is the
second most common isotype in the mucosa after IgA. In responses against thymus
independent (TI) antigens like polysaccharides, there is a strong induction of antigen
specific IgM. It is also the major isotype of so called “natural antibodies” which are
produced without being part of a humoral response against a specific antigen. Generally
the strength of the antigen-antibody association, the affinity, of IgM antibodies is low.
However, since IgM is secreted as a pentamer it can compensate for the low affinity by
carrying a total of ten possible binding sites. The increase in the possible number of
antigen-antibody interactions enhances the total affinity, the avidity. The pentamer form
enables the IgM molecule able to bind with high avidity to antigens that have the same
epitope in multiple copies. IgM is also the most efficient antibody class in binding and
activating complement, enabling it to opsonize or destroy targets by lysis.
IgD
IgD is only secreted to a minor extent; instead it is expressed together with IgM on the
cell surface, for example on mature naïve peripheral B cells and on follicular B cells.
IgD and IgM are the only Ig classes that can be co-expressed. Studies of mice lacking
IgD have not been able to reveal its exact immunological role. However, the fact that
IgD is present in all mammals and is well conserved among species suggests that it has
a distinct purpose.
IgG
High titers of IgG are characteristic of a secondary immune response, and it is also the
most common Ig class in blood and lymph. The membrane IgG, and IgE, have a longer
cytoplasmatic tail than the other immunoglobulin classes, which seems to enhance the
efficiency of memory responses [Achnatz et al 1997; Kaisho et al 1997; Martin and
Goodnow 2002]. The IgG class consists of four subclasses, IgG1, IgG2a, IgG2b and
IgG3, in mice, and IgG1, IgG2, IgG3 and IgG4, in humans. Responses against different
- 13 -
types of antigens tend to preferentially use different IgG subclasses, anti-carbohydrate
specificities tend to be IgG2b and IgG3 in mice [Ivars et al 1983; Perlmutter et al 1978;
Slack et al 1980] and IgG2 in human [Barrett and Ayoub 1986; Riesen et al 1976;
Yount et al 1968]. Responses against proteins induce IgG1 [Riesen et al 1976; Scott and
Fleischman 1982] and anti-viral responses IgG2a [Coutelier et al 1987] in mouse, while
in man anti-protein and anti-viral antibodies often are IgG1 and IgG3 [reviewed in
Papadea and Check 1989; Skvaril and Schilt 1984]. The effector functions of the IgG
subclasses also differ, complement is most efficiently fixed by IgG2a and IgG2b [Ey et
al 1980; Neuberger and Rajewsky 1981] in mouse and IgG1 and IgG3 in human
[Bruggemann et al 1987; Dangl et al 1988].
IgA
The daily production of IgA is greater than the production of all other Ig classes
combined. IgA is the major Ig class in secretions such as saliva, mucus, gastric fluids
and tears. Secretory IgA coats all body surfaces except skin and is thus an important
first line of defense. IgA is also the major Ig in colostrum and breast milk, thereby
providing the neonate with a defense against intestinal pathogens. Secretory IgA is
polymeric, while serum IgA is predominantly monomeric. Serum and secreted IgA
originate from separate pools of B cells. Whereas secreted IgA is produced by plasma
cells in specialized sites of the respiratory, urogenital, gastrointestinal and mammary
tissue, the serum IgA is produced in BM, lymph nodes and the spleen [Craig et al 1971;
Fagarasan et al 2001; Kutteh et al 1982]. The protection mediated by secreted IgA
consists of different functions. IgA binding and cross-linking pathogens prevent the
uptake of the pathogens across the epithelia and thereby facilitate the pathogen removal
in the mucus excretions. Furthermore, IgA-antigen complexes can be transported across
the epithelia into the lumen and at the stromal side IgA-antigen complexes can be
eliminated by phagocytes expressing receptors for IgA, FcαR [reviewed in van Egmond
et al 2001]. It has been shown that mucosal IgA responses can be induced in a T cell
independent manner and it was suggested to be a part of an evolutionary old form of
specific immune defense [Macperson et al 2000].
IgE
IgE is mainly produced by plasma cells in the lung and skin. IgE antibodies can be
bound to FcεRI on mast cells and basophils. Multivalent antigens can then cross-link the
bound IgE and thereby indirectly cross-link the FcεRI molecules. This will induce
degranulation and cytokine production by mast cells and basophils. While this type of
response can be important for anti-parasite responses, it also causes allergic reactions in
predisposed individuals.
- 14 -
Fc receptors
One type of receptors that is involved in antibody recognition is the Fc receptors
[reviewed in Daëron 1997; reviewed in Ravetch and Kinet 1991]. They recognize and
bind the Fc part of antibodies. Fc receptors exist for all antibody classes. They can be
divided into receptors that can promote or inhibit cell activation as well as receptors that
are involved in the transport of Ig through epithelia. Receptors that mediate antibody
transportation are the neonatal Fc receptor for IgG, which facilitates transport of
maternal IgG into the fetal circulation, and the polymeric IgA and IgM receptor.
Signaling through Fc receptors induce a great variety of actions depending on the cell
type carrying the receptor. Examples are the release of inflammatory mediators,
cytolysis and mediation of phagocytosis as well as a negative feedback regulation of B
cells.
Activation signals
B cell antigen receptors exist as a complex between membrane-bound Ig and at least
two other transmembrane polypeptides, Ig-α and Ig-β. Ig-α and Ig-β both have a single
external Ig-like domain, a transmembrane region and a cytoplasmatic tail which
interacts with signaling components [reviewed in Reth and Wienands 1997]. The
cytoplasmatic tail carries a sequence with homology to other leukocyte receptors, the
immunoreceptor tyrosin-based activation motif (ITAM). Cross-linking of receptor
molecules by oligomeric or multimeric antigens will trigger signaling through the BCR
(figure 4). Cross-linking will increase phosphorylation of specific proteins, and this
phosphorylation leads to altered activity of these proteins or to assembly of signaling
complexes. The initial event is phosphorylation of the ITAMs of Ig-α and Ig-β by Scr
family tyrosine kinases (Blk, Lyn, Fyn), which leads to the recruitment of Syk to the
receptor complex. When bound to a phosphorylated ITAM, Syk itself becomes
phosphorylated, which upregulates its activity. The activated tyrosine kinases will
trigger complex series of signaling events. Important signaling pathways are mediated
by phospholipase Cγ2 (PLCγ2), phosphatidylinositol 3-kinase (PI3K) and activation of
Ras.
antigen
BCR
Ig-α/β
Lyn
PLCγγ2
Btk
P
P
Ras
Figure 4.
BCR mediated signaling pathways.
Phosphorylation of the Ig-α/β ITAMs by
Scr family kinases such as Lyn recruits
Syk. After phosphorylation BLNK attracts
Btk and PLCγ2, which leads to the
activation of PLCγ2. Two other important
pathways is also initiated, the Ras pathway
and the PI3K pathway.
The figure is based upon figures in Gold
2002; Niiro and Clark 2002; Wang and
Clark 2003.
Syk
BLNK
PI3K
- 15 -
The activation of PLCγ2 seems to be dependent on Syk as well as Btk. Phosphorylation
of B cell linker protein (BLNK), probably by Syk, provides docking sites for Btk as
well as PLCγ2. When brought together, PLCγ2 is activated by Btk [reviewed in
Kurosaki et al 2000]. The PLCγ2 pathway will eventually lead to activation of the
transcription factor CREB (cyclic adenosine monophosphate response element binding
protein) [Xie and Rothstein 1995; Xie et al 1996] as well as the NFAT family
[Venkataraman et al 1994], Ets-1 [Fisher et al 1991], NF-κB and ATF-2 [Dolmetsch et
al 1997].
Co-receptors
An important aspect of BCR signaling is the modulation of the signal by so called coreceptors. These are cell surface receptors able to detect different elements of the
immune system and, when activated, to adjust the BCR signaling and regulate the
cellular response. Two important co-receptors for BCR signaling are the promoting
complement receptor CR2, and the inhibiting FcγRIIB [reviewed in Buhl and Cambier
1997]. Thus, if an antigen has been recognized by the complement system it will
enhance the B cell response, while high levels of antigen specific IgG will be an
indicator that no further antibody response is needed and will lead to the antibody
production being “turned off”.
Complement receptor 2
The complement system is a complex system of proteolytic enzymes, regulatory
proteins and surface receptors. It can on its own, as well as through interaction with
antibodies, mediate lysis of microbes. B cells express two receptors for complement
components, the complement receptors 1 and 2 (CR1 and CR2). CR2 enhances B cell
activation, and is primarily expressed on B and follicular dendritic cells (FDCs). On the
B cell, the CR2 can be found either in complex with CR1 or with CD19 and TAPA-1
[Matsumoto et al 1991]. CD19 can also be found associated with the BCR but at low
stoichiometry [Carter et al 1997]. Compared to cross-linking of BCR alone, crosslinking of CR2 or CD19 together with the BCR leads to synergistic PIP2 hydrolysis and
enhanced B cell proliferation [Carter and Fearon 1992; Carter et al 1988]. The
importance of CD19 and CR2 for the humoral response is apparent in mice deficient for
these receptors. Mice lacking CR2 or CD19 have a decreased response against thymusdependent (TD) antigens [Ahearn et al 1996; Croix et al 1996; Engel et al 1995; Molina
et al 1996; Rickert et al 1995]. Interestingly, the response against TI-2 antigens in these
mice is unaffected or increased. So although the CR2/CD19/TAPA-1 complex is an
important enhancer of the humoral response it does not appear to be essential for B cell
activation. The enhancement of the response is thought to be mediated through the
cytoplasmatic tail of CD19. When phosphorylated, the tail is recognized by PI3K
[Tuveson et al 1993] and Vav [Weng et al 1994]. CD19 can also associate to some
- 16 -
extent with Lyn and Fyn [Uckun et al 1993; van Noesel et al 1993], which could
promote the initial event of ITAM phophorylation.
FcγRIIB
Another co-receptor for B cells is the FcγRIIB, which is co-ligated with the BCR when
the antigen has the form of a soluble immune complex. FcγRIIB has low affinity for
IgG. Murine FcγRIIB binds IgG1, IgG2a and IgG2b subclasses but not IgG3
[Weinshank et al 1988]. The cytoplasmatic tail of FcγRIIB carries an immunoreceptor
tyrosine-based inhibitory motif (ITIM) which mediates a negative signal to the B cell,
possibly by inducing dephosphorylation of CD19 [Hippen et al 1997]. Examples of the
inhibition are decreased B cell proliferation [Phillips and Parker 1984] and antibody
secretion [Phillips and Parker 1984; Phillips and Parker 1985].
B cell responses against TD antigens
Different types of antigens induce humoral responses through different mechanisms.
When recognizing antigens such as proteins, B cells need co-stimulatory signals
provided by TH cells to be able to elicit a response. This type of antigen is called TD
antigens. Naïve B cells circulate through blood, lymph nodes and spleen until they
encounter antigen. This often happens in the T cell area in the spleen or lymph nodes, to
which antigen is brought by macrophages and dendritic cells. After encountering the
specific antigen, the B cell will stop migrating and will linger in the T cell zone
[reviewed in Kelsoe and Zheng 1993; Liu 1997]. B cells specific for the antigen will be
able to internalize the antigen via the BCR complex. The antigen is then processed and
presented on MHC class II molecules on the cell surface. In this area, naïve antigenspecific T cells can be activated, probably by antigen presenting interdigitating dendritic
cells. Upon antigen recognition activated T cells can form stable conjugates with the
antigen-presenting B cells, and deliver both receptor mediated and soluble stimulatory
signals to the B cells. This induces proliferation and terminal differentiation of the B
cells to plasma cells. These plasma cells produce the early wave of antibodies generated
after antigen challenge [reviewed in MacLennan et al 2003]. Some activated B cells do
not undergo terminal differentiation, but will instead migrate into the follicular region
and initiate the germinal center (GC) reaction. Though the complete requirement of the
signals needed for differentiation either into plasma cells or GC B cells is unknown, the
signaling via CD40 on the B cell induced CD40L on the T cell seems to be required for
GC formation [Foy et al 1994; Kawabe et al 1994].
B cell responses against TI antigens
Not all antigens need T cell help to activate B cells. This group of antigen is called TI,
and is further divided in to TI-1 and TI-2 type of antigens. The subdivision of TI-1 and
TI-2 antigens originates in their ability or inability to give rise to a humoral response in
XID (X-linked immunodeficiency) mice. These mice have lost the function of the
- 17 -
tyrosine kinase Btk, which is involved in BCR signal transduction. XID mice can
respond against TD and TI-1 antigen but not to polysaccharides, the classical group of
TI-2 antigens [Amsbaugh et al 1972; reviewed in Scher 1982]. The most striking feature
of the TI-1 antigens is their mitogenic properties. They are polyclonal B cell activators
that can induce differentiation of B cells into plasma cells regardless of their antigen
specificity. Many TI-1 are molecules found in bacterial cell walls, e.g.
lipopolysaccharide (LPS), peptidoglycan, and lipoprotein. The responses against LPS
have been studied extensively and it has been shown that B cells in mice carry a specific
receptor capable of recognizing LPS, the Toll like receptor (TLR) 4 [Hoshino et al
1999; Poltorak et al 1998].
TI-2 antigens are characterized by their repetitive structure, e.g. polysaccharides, and
some are capable of polyclonally activate B cells, e.g. dextran and levan [reviewed in
Coutinho et al 1974; reviewed in Coutinho and Möller 1975]. This type of antigens can
be found both as capsular polysaccharides in bacterial cell walls and on viral particles.
The importance of the repetitive structure for the TI-2 response has been shown by
using haptenated polyacrylamide as antigen. To elicit a strong in vivo TI antibody
response the molecule had to have a minimum of 12-16 haptens with proper spacing
[Dintzis et al 1976], suggesting that TI-2 antigens need to be able to efficiently crosslink BCRs. Although not dependent on T cell help, the TI-2 response is supported by T
cells [Endres et al 1983; Letvin et al 1981; Mond et al 1980; Wood et al 1982]. CD40
ligation [Dullforce et al 1998; Garcia de Vinuesa et al 1999; Snapper et al 1995] as well
as different cytokines [reviewed in Mond et al 1995, reviewed in Snapper and Mond
1996; reviewed in Vos et al 2000] improve the response. The source for these cytokines
during the TI response is unknown, both bystander responding T cells as well as NK
cells could be producers, and possibly also B cells.
The cross-linking of BCRs by TI-2 antigen is considered to be a first step of B cell
activation needing additional stimuli to generate a humoral response [Coutinho and
Möller 1974; reviewed in Möller et al 1991; reviewed in Vos et al 2000]. The BCR
cross-linking is thought to give a first signal as a result of the formation of small
clusters with cross-linked BCRs that induce signaling cascades [review in Vos et al
2000]. A more passive role of the BCR has also been suggested, where it would serve to
focus the antigen on the cell surface, thereby enabling pathogen-associated molecular
patterns to be recognized by pattern-recognition receptors which activates the B cell
[Alarcon-Riquelme and Möller 1990; Möller 1999; reviewed in Möller et al 1991]. TLR
receptors, capable of recognizing a wide variety of pathogen-associated molecules
[reviewed in Takeda et al 2003], are one type of receptors that could mediate a second
activating signal. LPS which is recognized by TLR-4 has proven to enhance TI-2
responses in vivo [Zhang et al 1988] as well as in vitro [Hosokawa et al 1989].
Furthermore, many TI-2 antigens can fix complement and thus induce enhancement of
- 18 -
the BCR signal by recruiting and activating the CR2/CD19/TAPA-1 complex [reviewed
in Carroll 1998].
Affinity maturation
As the immune response matures there is a shift in antibody repertoire [Berek et al
1991; Foote and Milstein 1991], the antigen-specific antibodies are of higher affinity
than early in the response and other Ig classes are used. This phenomenon is called
affinity maturation, and it mainly takes place in the environment of the GC. Somatic
hypermutation (SHM) is a process that introduces changes in the Ig genes and thus in
the ability of the Ig to bind antigen. During the hypermutation process, mutations will
be incorporated with a rate around 10-3/base pair/cell generation [reviewed in Allen et al
1987; Berek et al 1991; McKean et al 1984]. It has been calculated that with an
approximate length of the V region of 300 nucleotides, one nucleotide exchange will
take place in the VH and in the VL chain every third division.
The hypermutation target sequence, the mutation domain, spans over approximately
1500 nucleotides [reviewed in Wagner and Neuberger 1996]. Nucleotide substitutions
are seen only in rearranged V regions [Gorski et al 1983; Roes et al 1989; Weber et al
1991] and their flanking sequences [Lebecque and Gearhart 1990; Motoyama et al
1994; Rothenfluh et al 1993; Weber et al 1991]. The sequence of the V region is not
important for marking it a target for hypermutation. It has been shown that replacing the
gene with other sequences does not affect the introduction of mutations in the sequence
[Azuma et al 1993; reviewed in Storb et al 1996; Tumas-Brundage and Manser 1997;
Yelamos et al 1995]. To assess the probability of a base to be replaced, mutation
patterns have been studied in sequences not under selection pressure [Betz et al 1993;
Dörner et al 1997; Hackett et al 1990]. Transitions (A G and C T) are more frequent
than transversions (A C/T, G C/T, T A/G and C A/G), and there is a strand bias
for the coding strand. Some triplets are also more frequent targets, hot spots, than other,
mutational cold spots [Smith et al 1996; reviewed in Wagner and Neuberger 1996].
These hot spots often coincide with the CDRs [Betz et al 1993; Reynaud et al 1995].
→
→
→
↔
→
↔
The exact mechanism by which the mutations are introduced is not known. Some
observations suggest that it is a form of error-prone DNA repair mechanism: 1) the
dependence on an active promotor [Peters and Storb 1996; Tumas-Brundage and
Manser 1997], 2) the hypermutation domain starts downstream of the promotor region
[Lebecque and Gearhart 1990; Rothenfluh et al 1993; Weber et al 1991], 3) the
transcription enhancer sequences are required for hypermutation [Betz et al 1994], and
4) there is a strand bias [reviewed in Wagner and Neuberger 1996].
- 19 -
Recently it has been shown that the hypermutation process requires the expression of
activation-induced deaminase (AID), and it has been suggested that AID initiates SHM
by cytidine deamination of DNA [Petersen-Mahrt et al 2002]. Interestingly, mutations
of the AID gene lead to deficiencies in class switch recombination as well as in SHM
and gene conversion indicating that there is a common mechanistic link in these
processes [Arakawa et al 2002; Harris et al 2002; Muramatsu et al 2000; Revy et al
2000] (figure 5).
Class switch
VDJ E
Sµ Cµ Cδ
SHM
V region
Sx Cx
5'
3'
Introduction of
DNA breaks
Introduction of
DNA breaks
Cµ
Cδ
5'
3'
AID
Sµ
VDJ E
Sx
Cx
DNA repair
Cµ
Mutations by
error-prone
DNA synthesis
dependent
DNA repair
Cδ
VDJ E Sµ/x Cx
5'
3'
Sµ/x
Figure 5.
Ig class switch and SHM. A schematic model of the two mechanism. Nicking of
the switch (S) region and the V region is thought to be dependant on AID. The
repair during class switch will result in nonhomologous recombination and excision
of the intervening DNA. During SHM the DNA breaks will be filled by an error
prone DNA synthesis and the mutations will become fixed after DNA repair.
The figure is based upon figures in following papers, Diaz and Casali 2002; Kenter
2003; Kinoshita and Honjo 2001.
- 20 -
Class switch
In order to elicit an optimal antibody response, the B cells also have the ability to alter
the antibody class used in the response by exchanging the CH regions. Necessary for
class switching to take place is DNA-synthesis and transcription of the germline form of
the CH gene to which switching will occur, in addition to undefined processes that affect
the activity of a switch recombinase. The major mechanism for class switching includes
looping out and deletion of CH genes by nonhomologus recombination [reviewed in
Honjo et al 2002]. Switching typically occurs in sequences with limited or no homology
within so called “switch regions”. Switch regions act as binding or assembly sites and
are located 5´ of each CH gene except Cδ. Different stimuli are important for the
induction of class switch. Examples are the cytokines interferon-γ, and IL-10, often
described as “switch factors” [reviewed in Snapper et al 1997; reviewed in Stavnezer
1996]. They have the ability to induce transcription of the germline form for the isotype
to which switching will occur. Another group of stimuli, including LPS, cross-linking of
membrane Ig or CD40, acts in a more unspecific way to induce DNA-synthesis, Ig
secretion and class switch. IL-4 has the ability to induce both types of effects.
The germinal center reaction
When challenged with a TD antigen the major site for B cell activation is the
periarteriolar lymphoid sheath (PALS). The activated B cells will form foci of
proliferating cells near the arteriole [Jacob et al 1991a]. These B cells can have two
different fates, either they differentiate into plasma cells, or they will migrate into the
primary follicle and start the GC formation. The classical GC is divided into a dark and
a light zone. The dark zone consists of B cells called centroblasts that are proliferating
vigoursly. The light zone B cells, the centrocytes, are nondividing and in close contact
with a network of FDCs. The FDCs can present antigen to the B cells in the form of
immune complex bound to complement or Fc receptors on the cell surface [reviewed in
van Nierop and de Groot 2002]. Competition among the B cells for the bound antigen is
thought to create the selection of high affinity B cells. Those B cells that are not
selected will die by apoptosis, and the debris engulfed by tingible body macrophages at
the border between the dark and light zone. Some T cells can bee seen in the GCs, these
are antigen specific [Fuller et al 1993] mainly CD4+ and are found in the light zone
[reviewed in Kelsoe 1995].
The first clusters of proliferating GC B cells can be seen in the primary follicles three to
four days after antigen challenge. In the follicles the B cells will proliferate rapidly and
form a GC [reviewed in MacLennan and Gray 1986; Nieuwenhuis and Opstelten 1984].
The cell cycle of a centroblast can be as short as 12 to 6 hours [Fliedner et al 1964;
reviewed in MacLennan et al 1991; Zaitoun 1980]. By day 8 the centroblasts will fill
the FDC network. The naïve IgD+IgM+ B cells that occupied the primary follicles have
been pushed out and form what is called the mantel zone. The classical dark and light
- 21 -
zones of the GC will form over the next days. GC B cells will proliferate and
accumulate mutations in the dark zone, undergo selection in the light zone, leading
either to apoptosis, differentiation into memory or plasma cells or re-entry into the dark
zone (figure 6). With time, the number of centroblasts will decrease and around day 15
the GC is mainly occupied by centrocytes, and approximately three weeks after
antigenic challenge the GC reaction has faded [reviewed in Kosco-Vilbois et al 1997;
Liu et al 1991].
The proliferating B cells in the GC will include mutations in their Ig genes and will thus
give rise to daughter cells which carry variants of the original receptor [Jacob et al
1991b; Ziegner et al 1994], variants that have a possible different binding capacity.
These cells are selected for the highest affinity to the antigen and can after exiting the
GC differentiate into memory or plasma cells [Berek et al 1991; Liu et al 1996;
McHeyzer-Williams et al 1993; Smith et al 1997; Ziegner et al 1994;].
Dark zone
Light zone
• proliferation
• SHM
• selection
†
• apoptosis
memory
• re-entry to the dark zone
plasma cell
Figure 6.
The GC reaction. Antigen
activated B cells can be induced to
form a GC. In the dark zone
centroblast undergo rapid
proliferation and SHM. When
entering the light zone these cells
will compete for antigen on FDCs
and receive stimulatory or apoptotic
signals from GC T cells. The
centrocytes can either be promoted
leave the GC and further
differentiate into memory and
plasma cells, re-enter the dark zone
or undergo apoptosis.
Plasma cell differentiation
During a humoral immune response two highly differentiated cell types develop, plasma
cells which produce and secrete antibodies and memory cells that will facilitate a more
rapid secondary response of higher affinity. It is not clear which signals decide if a B
cell should leave the GC. Though not firmly supported by experimental evidence, it has
been proposed that signaling via high-affinity Ig would end the process of affinity
maturation [reviewed in Calame 2001]. The signals governing the decision between
plasma cell and memory cell fate is also unclear. The results from one study suggest that
plasma cell fate would be favored by expression of high-affinity antibodies [Smith et al
1997]. In vitro studies have also shown that co-stimulation via CD40 favors a memory
phenotype, whereas in the absence of CD40L, IL-2 and IL-10 induce plasma cell
- 22 -
differentiation [Arpin et al 1995]. IL-10 can also interrupt memory cell expansion
[Choe and Choi 1998], while IL-4 promotes formation of memory B cells [Zhang et al
2001]. Furthermore, the formation of plasma cells is suggested to be promoted by
signaling via OX40L [Stuber and Strober 1996].
Plasma cells are the terminally differentiated B cells. They do not divide and during
their life time, which can span between days up to months, they produce and secrete Ig.
The phenotype of the cell changes to meet the new demands. The cytoplasmic
compartment increases in size, and it contains large amounts of rough endoplasmic
reticulum and secretory vacuoles. Various surface molecules are down-regulated when
the B cell differentiates into a plasma cell, for example MCH class II, B220, CD19,
CD21 and CD22. Accordingly, the levels of different transcription factors will change
in the cell. Some transcription factors will decrease such as B cell lineage-specific
activator (BSAP), CIITA, early B cell factor (EBF) and Bcl-6, while the expression of
others, like B lymphocyte-induced maturation protein I (Blimp-1), interferon-regulatory
factor 4 (IRF4) and X box-binding protein 1 (XBP-1), are increased in plasma cells
[reviewed in Calame et al 2003]. XBP-1 is needed for plasma cell differentiation, but
not in earlier steps of B cell development [Reimold et al 2001]. It is expressed
ubiquitously; however, XBP-1 is induced in activated B cells and is strongly expressed
in plasma cells [Reimold et al 2001]. It is negatively regulated by BSAP [Reimold et al
1996], a transcription factor that decreases during differentiation to plasma cells
[Barberis et al 1990; Rinkenberger et al 1996], which could in part explain the increase
in XBP-1 during this stage. IRF4 is expressed mainly in lymphoid cells, and is
increased in plasma cells and a plasma cell-like subset of GC cells [Falini et al 2000].
Mice lacking IRF4 have severely reduced serum Ig levels, at least a 99% reduction, and
fail to produce antibodies in response to antigen challenge [Mittrücker et al 1997].
Blimp-1 was found as a gene being induced in IL-2 and IL-5 driven differentiation of
the cell line BCL1 into a plasma cell state [Turner et al 1994], and was later also
observed in splenocytes [Schliephake and Schimpl 1996]. Blimp-1 has been shown in
vivo to be expressed in all plasma cells, both in TI and TD responses and in secondary
responses [Angelin-Duclos et al 2000], and recently it was shown that Blimp-1 is
required for plasma cell differentiation [Shapiro-Shelef et al 2003]. Blimp-1 acts as a
transcriptional repressor of the c-myc [Lin et al 1997], CIITA genes [Piskurich et al
2000] and Pax5 [Lin et al 2002]. c-Myc is needed for proliferation and cell growth, and
the repression of its transcription can explain the non-dividing state of terminally
differentiated plasma cells. Likewise, the repression of CIITA provides an explanation
for the down-regulation of MHC class II expression in plasma cells. The repression of
Pax5, coding for BSAP, is probably important for triggering the plasma cell
development. BSAP is important in early stages of B cell development as well as for
isotype switching in GCs [reviewed in Michaelson et al 1996; reviewed in Morrison et
- 23 -
al 1998]. The formation of antibody secreting cells is dependent on down-regulation of
BSAP [Lin et al 2002; Usui et al 1997], probably due to BSAPs ability to repress XBP1 [Reimold et al 1996], J chain [Rinkenberger et al 1996] and Ig H chain [Singh and
Birshtein 1993] gene transcription. Blimp-1 has also the ability to down-regulate Bcl-6,
a protein necessary for GC formation [Dent et al 1997; Ye et al 1997]. Bcl-6 can on the
other hand repress the gene encoding Blimp-1, Prdm1, [Shaffer et al 2000] creating a
feedback loop controlling the B cell fate. While Bcl-6 is expressed in a GC B cell,
Blimp-1 expression is repressed and the differentiation into plasma cell is blocked.
However, when Prdm1 is induced, Blimp-1 will be able to inhibit the actions of Bcl-6
and the B cell will terminally differentiate into a plasma cell (figure 7).
Pax5
XBP-1
Bcl-6
Blimp-1
CIITA
IRF4
c-myc
Figure 7.
Transcriptional regulation of plasma
cell differentiation. XBP-1, IRF4 and
Blimp-1are important for plasma cell
differentiation. Blimp-1 negative
regulates Bcl-6 and Pax5, both
important for controlling the GC B cell
stage, as well as CIITA and c-myc.
Terminal differentiation
• Ig production
• stop in cell cycle
• change in surface proteins
Death and survival of mature B cells
Differentiation into long-lived mature B cells requires signaling by B cell activating
factor (BAFF) through BAFF receptor (BAFF-R) [reviewed in Laâbi et al 2001;
Schneider and Tschopp 2003]. BAFF deficient mice have normal numbers of early B
cell stages but lack mature B cells [Gross et al 2001; Schiemann et al 2001], and mice
expressing a BAFF transgene accumulated immature and mature B cells, had increased
Ig levels and showed signs of autoimmune disease [Gross et al 2000; Khare et al 2000;
Mackay et al 1999]. BAFF seams to support B cell differentiation and survival through
regulating expression of Bcl-2 family members [Do et al 2000; Hsu et al 2002].
Furthermore, BCR signaling is crucial for B cell survival, mature B cells that loose their
expression of the BCR dies rapidly [Lam et al 1997].
The negative selection in BM and spleen will result in cells either being removed by
apoptosis, rendered anergic or induced to undergo receptor editing [reviewed in
- 24 -
Nemazee 2000]. Whether removal of auto-reactive cells is induced via the death
receptor Fas is under debate [Rathmell and Goodnow 1994; Rubio et al 1996; reviewed
in van Eijk et al 2001a], but is reported to be blocked by Bcl-2 and Bcl-XL [Fang et al
1998; Hartley et al 1993; Lang et al 1997; Nisitani et al 1993]. Loss of the Bcl-2 family
member Bim has been shown to block apoptosis of autoreactive B cells, normally
released Bim interacts with Bcl-2 and inhibits its anti-apoptotic function [Enders et al
2003].
Activation of B cells leading to proliferation and differentiation requires signals through
the BCR and co-stimulatory receptors. CD40L [reviewed in van Kooten and
Banchereau 1997], BAFF [Moore et al 1999; Schneider et al 1999] and APRIL (a
proliferating inducing ligand) [Stein et al 2002] mediate important signals for the B cell.
Their receptors, CD40, TACI (transmembrane activator and CAML-interactor), BCMA
(B cell maturation antigen) and BAFF-R signal through the same group of transcription
factors, the Rel/NF-κB family [Berberich et al 1994; Kayagaki et al 2002; reviewed in
Laâbi et al 2001; Marsters et al 2000; Yan et al 2000]. This results in signaling induced
by CD40, BAFF and APRIL, apart from upregulating genes important for B cell
activation, also upregulates genes that promote survival, such as bcl-2, bcl-XL [Do et al
2000] and A1 [Kuss et al 1999]. The signals via BCR and costimulatory receptors work
in balance, solely CD40 ligation induces an upregulation of Fas which makes the cell
sensitive to apoptosis induced by Fas ligand, but BCR stimulation will protect the cell
[Rothstein et al 1995], probably through upregulation of the anti-apoptotic protein FLIP
(FLICE-inhibitory protein) [Wang et al 2000].
Antigen activated B cells can terminally differentiate into antibody secreting cells in the
spleen or form GCs. The cells in the spleenic foci will eventually undergo apoptosis
plausibly due to a limited access to essential cytokines, a death that can be blocked by
Bcl-2 over-expression [Smith et al 1994]. When the centroblasts enter the light zone in
the GC they compete for antigen in form of immune complexes presented by FDCs.
This leads to a selection of high affinity B cells, which are able to interact with the
FDC. Interaction with FDC has been shown to protect from Fas mediated apoptosis
[Koopman et al 1997; Schwarz et al 1999; van Eijk et al 2001b]. Cells with low affinity
receptors are presumed to undergo apoptosis through neglect, and this apoptosis can be
blocked by Bcl-2 [Smith et al 2000] or Bcl-XL [Takahashi et al 1999].
Another selection step that B cells must pass in the GC light zone is selection on antigen
specific TH cells. Antigen specific T cells present in the light zone can give signals to B
cells that both promote apoptosis and protect from it. Interaction between CD40 on the
B cell and CD40L on the T cell leads to up-regulation of Bcl-XL [Choi et al 1995; Wang
et al 1995] and of Fas [Garrone et al 1995; Schattner et al 1995]. However, signaling
through BCR will mediate rescue signals from Fas mediated apoptosis [Rothstein et al
- 25 -
1995]. Thus, B cells that do not bind antigen will be eliminated by T cells, which carry
the Fas ligand, through Fas induced apoptosis. Mice with a mutation in Fas, the lpr
mutation, develop a lymphoproliferative disease with autoreactive B cells that have
undergone SHM [Shlomchik et al 1987]. The exact role of Fas in GC selection is not
clear, although GC, memory and antibody forming cell populations have been reported
to be normal in lpr mice [Smith et al 1995], a more recent study have shown defects in
clonal selection and an accumulation of maturations in these mice [Takahashi et al
2001]. When an immune response ends, the expanded clones of effector cells must be
reduced in size. This process is dependent on Bim through an apoptotic pathway that is
blocked by Bcl-2. Both bcl-2 transgenic and Bim-deficient mice show a disturbed
immunological homeostasis with an accumulation of antibody forming cells [Bouillet et
al 1999; Smith et al 1994; Strasser et al 1991].
Humoral immunity
Humoral immunity is upheld by three different mechanisms. An immune response will
induce the formation of short lived plasma cells that are able to create a fast increase in
antibody levels. In a more long term perspective, specific antibodies are being produced
by long-lived plasma cells, mainly residing in the BM. In contrast to short lived plasma
cells with life spans measured in days these cells live for several months [Manz et al
1997; Slifka et al 1998]. However, these cell populations are terminally differentiated,
however, and are not able to maintain the antibody response for prolonged periods. True
long term memory is sustained by a heterogeneous group of cells called memory B
cells. The phenotypes of memory B cells are rather poorly defined. Most memory cells
express switched Ig isotypes, but also IgM memory cells can be found [Klein et al
1997]. Furthermore, memory B cells can be divided into two populations based on the
expression of B220 [Driver et al 2001; McHeyzer-Williams et al 2000]. Memory B
cells expressing B220 (B220+) have the highest potential of cellular differentiation and
proliferation capacity upon antigen re-challenge, while memory B cells lacking B220
(B220-) have the ability to self-replenish and are predisposed to form antibody secreting
cells. A model of linear differentiation has been proposed, where B220+ memory cells
give rise to B220- cells from which antibody secreting cells are generated [McHeyzerWilliams et al 2000].
The GC has since long been considered the main site for directing B cells to the
memory pathway, however, it does not seem to be absolutely required as knock-out
mice lacking GCs has been shown to have memory B cells [Toyama et al 2002]. After
leaving the GC, memory B cells can be divided into recirculating memory B cells and
cells localized in the marginal zone in the spleen and lymph nodes [Liu et al 1988; Liu
et al 1991; Liu et al 1996; Nieuwenhuis and Ford 1976; Oldfield et al 1988]. Since
marginal zone B cells are in regions where blood-borne antigens drain, they are fast
responding during secondary challenges. Rechallenge with an antigen leads to rapid and
- 26 -
massive differentiation of memory cells into blasts and plasma cells [reviewed in
MacLennan et al 1992; reviewed in van Rooijen 1990]. Repeated antigen challenges
can further increase the antibody affinity, which suggests that either the reactivated cells
are capable of undergoing further affinity maturation or that the newly recruited naïve B
cells must show higher affinity than the memory response to survive the GC process.
The need for restimulation with antigen for maintaining memory B cells is under debate.
Antigen can be stored for a long time as immune complexes trapped at the surface of
FDC [reviewed in Szakal et al 1989; Tew and Mandel 1979] and viruses can stay in
host tissues for years. In agreement with the notion that maintenance of memory need
antigen, it has been shown that transfer of memory B cells, in the absence of antigen,
leads to the loss of the memory B cell population [Gray and Skarvall 1988]. In contrast,
an experiment where the use of genetically modified mice enabled the specificity of the
memory B cells to be changed indicated that long-lived memory B cells could survive
without antigen [Maruyama et al 2000]. It has also been suggested that serological
memory is maintained through polyclonal activation of memory B cells [Bernasconi et
al 2002].
Humoral responses and aging
Aging affects the body and its functions, so also the immune system. Thymic
involution, decreased T cell responses, changes in cytokine expression of T cells and
accumulation of memory T cells have been considered to contribute to the decline of the
aging immune system [Knight 1995; reviewed in Miller 1996]. Humoral responses also
decline with age, both in quantity and quality. The titers of antibodies produced in
immune responses in aged individuals are often lower than in young adults [Burns et al
1990; reviewed in Miller 1991], and the binding capacity of these antibodies is also
lower [Doria et al 1978; Goidl et al 1976; Kishimoto et al 1976; Weksler et al 1978;
Zharhary et al 1977]. Possibly due to a changed function of aged TH cells, aging also
induces changes in the V gene usage [Nicoletti et al 1991, reviewed in Song et al 1997;
Yang et al 1996], leading to a change in B cell repertoire. The effect of aging is more
pronounced in TD immune responses than in TI response [Goidl et al 1976; Smith
1976; Weksler et al 1978]. It has been suggested that it is mainly due to deficiencies in
the TH cells [reviewed in Miller 1996; reviewed in Song et al 1997]. The formation of
GC, dependent on TH cells, is impaired in old mice [Miller and Kelsoe 1995; Szakal et
al 1990]. However, it has been suggested that the defective GC formation is not caused
by an aged T cell compartment, but instead to be due by age related changes of FDC
[Aydar et al 2003; Szakal et al 2002]. It is also likely that the efficiency of the GC
reaction is reduced by age, since a decrease in VH gene mutation in GC B cells has been
reported [Miller and Kelsoe 1995; Yang et al 1996].
- 27 -
Present study
Aim
The work presented in this thesis has dealt with how different factors such as age,
antigen type and defective apoptosis influence the humoral immune response. The
specific aims for each paper are as follows:
Paper I:
Aging affects both the B and T cell compartment. By studying the response against TI
and TD antigens we evaluated the effect of aging on humoral responses. In particular,
we examined what effect antigenic priming early in life had on humoral immune
responses in old individuals.
Paper II:
Mice immunized with the TI antigen dextran B512 (Dx) have a reduced IgG1 response
to later challenges with a TD form of Dx. We investigated if this phenomenon could be
induced by other TI antigens and also examined possible mechanisms contributing to
the low IgG1 response.
Paper III:
Certain TI antigens can induce the formation of GC, but in general the GC reaction is
unproductive. We wanted to see if this could be due to an early commitment to the
plasma cell fate by studying the expression of Blimp-1, a master regulator for plasma
cell differentiation, in GC induced by a TI antigen.
Paper IV:
For a productive GC reaction, apoptosis is though to have an important role. In this
paper we studied the effect on the accumulation of SHM in GC B cells from Peyer’s
patches in mice with disturbed apoptosis, namely lpr mice which carry a mutated
variant of the death receptor Fas, and in mice over-expressing Bcl-2, an anti-apoptotic
protein.
- 28 -
Methodology
This section describes briefly the methods used in the different experiments; they are
described in detail in each paper.
Mice
Several different mouse strains were used in these studies and they all shared a
C57BL/6 background. C57BL/6 is a strain that is a high responder against Dx
[Fernández and Möller 1977; Fernández et al 1979]. Except from wild type C57BL/6,
we also used BLκ6 mice (Paper II). These mice are the progeny from Lk6 transgenic
mice, which carry five copies of a gene construct of a mouse VκOx1-Jκ5 rearrangement
coupled to a rat constant κ chain [Meyer et al 1990], breed onto a C57BL/6 background.
The mice used in Paper IV with a defective apoptosis originated from B6.MRLTnfrsf6lpr (The Jackson Laboratory, Maine, USA), which carries a mutated gene coding
for the Fas protein, and the transgenic C57BL/6-Eµ-bcl-2-36 strain, which overexpresses human Bcl-2 on both B and T cell compartments [Strasser et al 1990]. These
two strains were crossed with the BLκ6 mice generating apoptosis deficient mice
carrying the Lk6 transgene called lpr/ox and bcl-2/ox respectively. In Paper II a
FcγRIIB knockout mice [Takai et al 1996] were used to study the importance of FcγR.
Nude mice (MB, Ry, Denmark), which are athymic due to a developmental failure of
the thymic anlage, and consequently lack mature T cells, were used in Paper III to
assess the contribution of the T cell compartment.
Immunizations, Antigens and Adjuvants
The animals were challenged with antigen by intraperitoneal immunization. Since Dx
B512 was used as a model antigen, most of the other antigens used were Dx hapten or
Dx protein conjugates. Chicken serum albumin was conjugated with Dx with a MW of
103 to obtain a TD form of Dx. TI forms of Dx carrying the haptens 2-phenyl oxazolone
(Ox), and dinitrophenyl (DNP) were generated by conjugating 4-ethoxymethylene-2phenyl-2-oxazolin-5-one, (Sigma-Aldrich Corp., St. Louis, MO, USA) and Nε-2,4DNP-L-Lysine, (Sigma-Aldrich Corp.) respectively to Dx with a MW of 2 x 106. Other
antigen used were a TD form of Ox, which was obtained by conjugating Ox to bovine
serum albumin (BSA), and BSA-DNP, which was obtained from Biosearch
Technologies (Novato, CA, USA). When antigen was administered with adjuvant the
adjuvant used was cholera toxin (CT) (List Biological Laboratories Inc., Campbell, CA,
USA). In paper II Dx specific antibodies originated from hybridoma cell lines were
administered.
ELISA
The levels of antigen specific antibodies were measured by ELISA. Briefly the plates
were coated with the desired antigen. Sera obtained from the immunized animals were
- 29 -
added in serial dilutions. Bound antibodies were detected using alkaline phosphatase
labeled antibodies specific for different mouse Ig isotypes, and p-nitrophenyl phosphate
substrate. The optical density was measured at 405 nm. In addition, ELISA was also
used to screen the BLκ6 offspring in the different breedings for the presence of rat Cκ.
More details on the different ELISAs can be found in the respective paper.
Estimating antibody affinity
In paper I the affinity of the antibodies was assessed by measuring the apparent
association constant aKa. The aKa value is defined as the reciprocal value of the
concentration of free ligand that gives a 50% inhibition of antibody binding to the
antigen coated plate. Bound antibodies were detected using antibodies specific for
mouse Igs labeled with alkaline phosphatase. The plates were developed using the
substrate p-nitrophenyl phosphate and the absorbance was measured at 405 nm.
Immunohistochemistry
Spleens taken from treated mice were frozen and stored in -70 to -85°C. Pieces of the
frozen spleens were embedded in Tissue Tek OCT compound (Miles, Elkhart, IN,
USA), cryosectioned (6µm), mounted on slides and stored. For staining, the slides were
allowed to dry before they were fixed in acetone. After the slides had dried and been
washed they were blocked using horse serum before adding the primary antibodies. The
slides were then washed and incubation with secondary reagents before a last wash and
mounting. The slides were then examined using a UV-microscope.
Generation of hybridoma cell lines
In paper II, mice were hyper-immunized with either Dx or CSA-Dx together with CT.
Spleens were taken day 3 after the third immunization and fused with the non-secreting
hybridoma cell line SP2/0 using polyethylene glycol solution Hybri-Max (SigmaAldrich Corp.) as fusing agent. The cultures were grown under the selection pressure of
HAT-media, Hybri-Max (Sigma-Aldrich Corp.). The antigen specificity of the clones
was determined by ELISA as described above.
Extraction of GC B cells from Peyer’s patches
A single cell suspension was prepared from Peyer’s patches dissected from the small
intestine of the experimental mice. After washing in cold PBS, the cell suspension was
filtered before a final wash in cold PBS. The cells were stained using FITC-conjugated
peanut agglutinin (PNA) (Sigma-Aldrich Sweden AB, Stockholm, Sweden), PNA being
a marker for GC B cells [Rose et al 1980], and phycoerythrin-conjugated anti-mouse
CD45R (B220) (Pharmingen, San Diego, CA, USA), a B cell marker. The lymphocyte
population was gated using the parameters of size and complexity in a Becton
+
high
Dickinson FACSVantage SE, and the B220 PNA cell population was purified by
- 30 -
sorting. This population corresponds to GC B cells. The sorted cells were centrifugated
and the pellets were stored at -70°C.
DNA and RNA isolation, RT-PCR amplification and sequencing
This part of the work was done by our collaborators in Spain, Elena Fernández
Mastache and África González-Fernández. Briefly, for analysis of the accumulation of
mutations in the VκOx1 gene (paper IV) DNA was extracted from the frozen cell pellets
obtained by cell sorting. The sequence of interest was amplified using PCR. The PCR
product was purified and re-amplified. The re-amplified product was cloned into
plasmid vector pBluescript+. Positive clones were expanded and the plasmid was
purified using QIAprep Spin Miniprep Kit (QIAGEN GmbH). The sequence reaction
was performed using the Dye terminator Cycle Sequencing kit (Beckman Coulter,
Fullerton, CA), and the products sequenced in an automated DNA sequencer (CEQTM
2000, Beckman Coulter). The sequences obtained were analysed using the Clustal X
(version 1.8) and the Genedoc (version 2.6.002) software.

For analysis of the VH gene usage (paper II) RNA was isolated from hybridoma cell
lines using the SV Total RNA Isolation System (Promega, Madison, WI, USA). cDNA
was generated using a primer specific for the constant µ region, and the reverse
transcriptase M-MLV (Promega). cDNA reaction mixture was amplified in a PCR
reaction using primers complementary to the JH and VH genes. The PCR product
purified and the sequence reaction and sequencing was carried out as mentioned above.
The program IgBlast (NCBI) (http://www.ncbi.nlm.nih.gov/igblast) was used to
determine the identity of the VH genes.
- 31 -
Results
Here follows a summary of the results obtained in the different papers. References to
figures and tables indicate figures and tables in each individual paper.
Paper I
Age is known to affect the function of the immune system. Both the B and T cell
compartment is affected, often leading to an impaired immune responsiveness in old
individuals. In this study we investigated the effect of aging on responses to TI and TD
antigens, and in particular we examined the effect of early life priming for responses in
old animals. When analyzing the response against TI Dx, we could see that the levels of
specific IgM after primary immunization were similar in young and old mice (fig 1),
indicating, in consistency with other data [Weksler et al 1978], that the B cell
compartment is not so affected by aging. Furthermore, though old mice immunized with
TI Dx generated approximately as many GC as young mice, the Dx binding capacity
was reduced in the GC of the old mice (fig 2). This suggests that the number of Dx
specific B cells is lower in these GC, since the IgM antibody affinity is similar in young
and old mice (table 2). When looking at TD Dx responses and the levels of Dx specific
IgG, the differences between young and old animals are more pronounced (fig 3), as
could be expected as a result of an impaired T cell function [reviewed in Song et al
1997; reviewed in Zheng et al 1997]. We were particularly interested in the effects
antigen priming in young mice had on the immune response in aged mice. In TD
responses we show that early life priming with CSA-Dx, together with CT, greatly
improves the IgG response of the aged mice against Dx (fig 3). It is known that priming
with TI Dx causes a reduced IgG response to later TD Dx responses [Sverremark and
Fernández 1998a]. We show in this paper that the reduction is long lasting, it is distinct
still one year after TI priming (fig 5) and that it also decrease the affinity of the Dx
specific IgG that is produced (table 3).
Conclusion: In agreement of earlier presented data, we show that the T cell
compartment is more affected by aging than the B cell compartment. Furthermore, we
highlight the importance of early life priming for later responses. We show that priming
can result in both an improvement of the response in the old mice and cause a reduced
response which is the case after TI priming.
Paper II
Knowing that TI Dx priming can have a major effect on later TD Dx responses we
investigated possible mechanisms behind this effect and if this reduction could be
induced by other TI antigen. Clonal exhaustion caused by activation in the absence of
memory formation, could possibly be one explanation for the effects of TI Dx priming.
The use of CT strongly improves the TI Dx response, indicating that either the
responding clones are not exhausted or that, in the presence of CT, other clones are
- 32 -
activated in the secondary response. We compared the VH chain usage in Dx specific
hybridomas generated from animals hyperimmunized with either TI or TD Dx together
with CT. The VHB512 gene was used in both TI and TD Dx specific responses (Table
1). It is the same gene that codes for a majority of antibodies in primary responses
against Dx [Fernández 1992]. Thus, we concluded that neither the use of CT, activation
by TI or TD forms of Dx, nor repeated immunizations seem to influence the gene usage
in the Dx specific response. Consequently, since there is no shift in gene usage, clonal
exhaustion does not seem to be contributing to the reduced TD responses induced by TI
Dx priming. To examine the effects of Ig dependent mechanisms on TD Dx responses
after TI Dx priming, we administered Dx specific antibodies to mice and challenged the
mice with CSA-Dx after a resting period. Treatment with Dx specific antibodies did not
lead to a reduction of Dx specific IgG1 in the TD response (fig 1). Furthermore, mice
lacking FcγRIIB were as affected by TI Dx priming as wild type mice (fig 2). These
results suggest that Ig dependent mechanisms, such as generation of anti-idiotypic
antibodies or negative signaling through Fc receptors, do not seem to be the cause to the
reduction of Dx specific IgG. We could also demonstrate that the negative effect of TI
priming is not exclusive to responses against Dx. Using the restricted hapten Ox, we
show that TI priming induced a reduction of IgG1 levels in later TD Ox responses. This
led us to propose that the reduced TD response after TI priming is due to a property of
the TI activation itself, presumably to directing the responding cells to a nonswitch/non-memory pathway.
Conclusion: In this paper we rule out clonal exhaustion and Ig dependent mechanisms
as main factors behind the reduced TD IgG1 responses induced by TI priming. We also
show that this effect can be directed to another molecule than Dx.
Paper III
We continued to examine differences between TI and TD responses, and in particular, if
the different types of antigens induce the responding cells to mature differently. The GC
is a specialized environment where affinity maturation, class switch and induction of
memory formation occur. While the formation of GC is typical of responses against TD
antigen some TI antigens such as Dx can also induce GC formation [de Vinuesa et al
2000; Sverremark and Fernández 1998b; Wang et al 1994]. However, the response
against TI Dx does not show signs of substantial affinity maturation or memory
formation. In this paper we investigated if this could be due to differences in expression
of the transcriptional repressor Blimp-1 in the GC. Blimp-1 expression drives the cell to
plasma cell maturation and regulates the expression of Bcl-6, needed for the formation
of GC, in a negative feedback loop. By staining spleen sections from immunized mice
with PNA and Blimp-1 specific antibodies, we show that GCs induced by TI antigen
express Blimp-1 earlier in the response (fig 1 and 2) and that the staining is stronger
than in TD induced GC (fig 3). Furthermore, in TD responses after TI priming the
- 33 -
Blimp-1 expression early in the formation of the GC resembles that seen in TI
responses, while the expression in more mature GC is similar to that in TD GC (fig 2
and 3).
Conclusions: GCs induced by TI antigen express Blimp-1 at earlier time-points and
have a higher staining intensity than TD induced GCs. TD GCs in mice that earlier have
been primed with TI antigen show a mixed pattern of Blimp-1 expression, early it
resembles more the TI induced GC but as the GC matures the expression becomes
similar to that in TD GCs. This suggests that the response is mixed and not simply a
suppressed TD response as thought before.
Paper IV
By using mice with deficiencies in the apoptotic pathways, mutated Fas or overexpression of Bcl-2, as well as carrying a mouse/rat Vκ gene construct, we studied the
effect of these deficiencies on the accumulation of SHM. GC B cells (PNAhigh B220+)
were isolated from Peyer´s patches and the mutations in the inserted Vκ genes were
studied. We found that the mice carrying the lpr mutation displayed a normal mutation
pattern, with regard to mutation of intrinsic hotspots (fig 3A). However, the lpr/ox mice
showed an increased frequency of T to A transversions (fig 2A). In addition, there was
an increase in the frequency of mutations (table 1) as well as in the incorporation of
deleterious mutations (table 3). In contrast, the over-expression of Bcl-2 showed a
decreased mutation frequency (table 1). Furthermore, these mice showed alteration in
the nucleotide substitutions, normally the A/T ration is close to 2, but these mice
showed no mutational bias for the mutation of A, A/T ratio 1.1 (table 2) and the G/C
ratio was of 0.6 instead of the expected 1. The mice over-expressing Bcl-2 also showed
a reduced number of mutations in the intrinsic hotspots (fig 3B, table 3).
Conclusion: We show that a mutated Fas does not lead to any major changes in the
characteristics of the SHM introduced, but contributes to an increase in the frequency of
mutations. In contrast, the over-expression of Bcl-2, leads to a different pattern of
mutations as well as a reduction of the overall number of mutations. This could be due
either to disturbed apoptosis or perhaps more likely to other effects of the overexpression. Over-expression of Bcl-2 is for example known to retard the cell cycle
progression [Mazel et al 1996; O’Reilly et al 1996], which could possibly allow for
repair of the mutated sequences.
- 34 -
Concluding discussion
Like all biological processes the induction of an immune response is shaped by a
combination of both internal and external factors. In this thesis I have investigated a
couple of different factors that have an impact on how the responses is induced and
developed, e.g. age, type of antigen and apoptosis.
In the GC where B cell clones are greatly expanded in the presence of a mutationmachinery, apoptosis plays an important role to weed out cells that potentially could be
dangerous or that have a low affinity for the antigen. Though it is not doubted that
apoptosis is important for selection in the GC the debate is ongoing with regard to
which apoptotic pathway is the most important. In our studies we could se that mice
deficient in Fas mediated apoptosis accumulated mutations in the GC B cells, an
accumulation that is mirrored in the phenotype of the lpr mice where there is an
accumulation of mutated B lymphocytes. Over-expression of Bcl-2 gave another
phenotype, a lower number of mutations and differences in nucleotide preferences
compared to wild type mice. This led us to conclude that impaired apoptosis caused by
the lpr mutation or over-expression of Bcl-2 affects the GC reaction. Furthermore, these
effects can be seen at different stages of the GC reaction.
The fact that age affects the efficiency of the immune system is known. In both very
young and very old immune responses are often weakened [reviewed in Castle 2000;
reviewed in Siegrist 2001]. A better understanding of why we can se these effects and
how to counteract them would of course give big humanitarian and economical benefits.
We show that antigen encountered early in life can greatly influence the response in an
aged individual, both in a positive and negative manner. The long lasting effects of
priming in young individuals, suggests a possible future development where vaccines
are constructed not only to give a desired protection, but also to maintain a strong
immune response in an aged individual.
One group of antigens that both very young and old individuals have difficulties to
respond against is polysaccharides. As these molecules can be found on bacterial cell
walls as well as viral capsules they constitute antigens that we are constantly challenged
with. The model antigen we have used is the polysaccharide Dx B512 from the bacteria
Leuconostoc mesenteroides. In common with other TI-2 antigens, the response against
Dx shows no sign of affinity maturation or formation of memory. In fact, secondary
responses are suppressed compared to primary ones. However, this suppression can be
abrogated by the use of CT as adjuvant. A peculiar effect that is seen after TI Dx
priming is that later responses against TD Dx have markedly reduced levels of IgG1. As
we found in our studies (paper I), this effect can be very long lasting. After ruling out
clonal exhaustion and Ig dependent mechanisms as possible mechanisms for inducing
this kind of unresponsiveness, we became increasingly convinced that the lack of switch
- 35 -
and memory in these responses is caused by properties of the activation induced by the
TI antigen.
In the past, differences between TI and TD responses have often been explained by the
lack of T cell help in the former. However, although certainly not without importance,
lack of T cell help might not be the only thing shaping responses against TI antigen.
Activation by TI antigen is thought to occur by cross-linking of the BCR coupled to a
second signal for example via pattern recognition receptors or complement receptors. TI
activation has been shown to induce a different phenotype of the responding B cells
than TD like activation [Cong et al 1991; Haas and Estes 2000; Wortis et al 1995],
suggesting that the signals induced by the respective activation lead to differences in the
maturation of the responding cells. When a B cell is activated during a TD response, it
is pictured that the maturation of the cell can proceed in two different directions. Either
it differentiates terminally and become a plasma cell or it can enter the germinal center
reaction characterized by the incorporation of SHM, class switch and memory
formation. As the responses against Dx largely lack class-switched antibodies and a
proper memory formation, we wanted to know if this was due to an early commitment
of the responding B cells to the plasma cell fate.
We investigated if GC induced by TI Dx differed from TD antigen induced GC with
regard to the expression of Blimp-1. Blimp-1 is the master regulator of terminal
differentiation of B cells into plasma cells. It also regulates the expression of Bcl-6, a
protein needed for GC formation, through a negative feedback loop. We found that TI
GCs express Blimp-1 earlier in the response. Our interpretation is that the responding B
cells are constantly recruited to a plasma cell fate, and leave the GC before SHM,
switch and induction of the memory pathway has occurred. It has been shown that BCR
signaling leads to degradation of Bcl-6 [Niu et al 1998]. It is possible that Dx can
induce a strong enough signal, either through the BCR or some other type of receptors,
such as pattern recognition receptors, to induce degradation of Bcl-6. Another
possibility is that the responding B cells fail to get co-stimulatory signals by TH cells
that would normally maintain or increase the levels of Bcl-6.
So, if activation with TI antigen drives the responding cells towards differentiation into
plasma cells, is that a good or a bad thing? With a TD antigen perspective the answer is
bad. Humoral responses should undergo affinity maturation to improve the affinity,
class switch to gain other functions for the antibody and memory to be able to respond
quicker the second time the antigen is encountered. However, one can take another
viewpoint. First the apparent lack of memory could be a lack of class switched memory
cells, there indications that TI antigen can induce an IgM memory [Hosokawa 1979;
Kolb et al 1993]. And considering that polysaccharides often are found in bacterial cell
walls, IgM might be a very important Ig class for the response. Due to the secretion of
- 36 -
IgM in form of a pentamer, it can bind with high avidity, and it is the most efficient
activator of complement of the Ig isotypes, making it able to mediate lysis of the
invading pathogen. So maybe it is time to look at TI responses as a different kind of
humoral responses instead of defective TD responses?
- 37 -
Acknowledgements
I would like to express my sincere gratitude to family, friends and colleges who have
helped and supported me in so many ways during the years. Especially I would like to
thank the following people:
Carmen Fernández, my supervisor, for your inspiration, caring and enthusiasm, and for
your ability to always find something positive even in the worst results.
Göran Möller, for accepting me as a student at the department.
The seniors, former and present, Manuchehr Abedi-Valugerdi, Klavs Berzins, Alf
Grandien, Eva Sverremark-Ekström, Marita Troye-Blomberg, for all the help and for
sharing your passion for and knowledge of different parts of immunology.
Peter and Hedvig Perlmann, you are an inspiration for us all.
Margareta Hagstedt, Lena Israelsson, Ann Sjölund, Gunilla Tillinger, Gelana Yadeta
for always helping me when I needed it.
Elena Fernández Mastache, África Gonzalez Fernández and Marta Sánchez, my
Spanish collaborators, for your hard work and nice discussions.
My roommates, Monika Hansson, for always being ready to help me and to discuss
immunology, dogs and all other things over a cup of coffee! Anna Tjärnlund for all the
laughs and being such a nice person, Masahi Hayano, for your humor and for putting up
with all the chatty girls.
My former roommate, Eva Nordström, without you I would never have tested green
milk!
To the other students that have come and gone during my time at the department, Ariane
Rodríguez-Muñoz, Anna-Karin Larsson, Mounira Djerbi, Valentina Screpanti, Cecilia
Rietz, Nina-Maria Vasconcelos, Elisabeth Hugosson, Caroline Ekberg, Anki Häglind,
Gun Jönsson, Qasi Kaleda Rahman, Salah Eldin Farouk, Ahmed Bolad, Manijeh Vafa,
Shiva S. Esfahani, Jacob Minang, Halima Balugon, Ben Adu Gyan, Susanne
Gabrielsson for being good friends both in and out-side the lab.
Izaura Roos, the one that got away…
Pia Lundell, Eva Nygren and Solveig Sundberg for your work in the animal house
taking care of all my mice.
- 38 -
Mamma och pappa för all den kärlek och det stöd som gjort mig till är den jag är!
Ulf, Björn och Leif, världens bästa bröder som har finslipat min argumentationsteknik
till det yttersta.
Mormor och morfar som oroligt sett mig fara kors och tvärs över världen. Men jag vet,
det är bäst hemma i Norrland!
Ronny och Bruno, för goda middagar och gott sällskap.
Sixtenssons, för att ni tagit emot mig med öppna armar och hjälpt till när det behövts.
Alla mina vänner, som haft tålamod med mig när jag bara haft jobb i huvudet, och som
jag tillbringat så många roliga timmar med i den verkliga världen (och nog minst lika
många i någon overklig…).
Tillsist,
Magnus, mitt hjärtas kär, för att du alltid finns där när jag behöver dig. Och för att du
ibland vet bättre än jag själv vad jag behöver!
- 39 -
References
Achatz, G., Nitschle, L., Lamers, MC., Effect of transmembrane and cytoplamic
domains of IgE on the IgE response. Science. 1997.276:409-411
Ahearn, JM., Fisher, MB., Croix, D., Goerg, S., Ma, M., Xia, J., Zhou, X., Howard,
RG., Rothstein, TL., Carroll, MC., Disruption of the Cr2 locus results in a reduction in
B-1a cells and in an impaired B cell response to T-dependent antigen. Immunity.
1996.4:251-262
Alarcon, B., Gil, D., Delgado, P., Schamel, WW., Initiation of TCR signaling:
regulation within CD3 dimers. Immunol. Rev. 2003.191:38-46
Alarcon-Riquelme, M., Möller, G., Thymus-independent B-cell activation: passively
incorporated membrane antibodies serve to focus the antigen but cannot transmit
activation signals. Scand. J. Immunol. 1990.31:423-428
Allen, D., Cumano, A., Dildrop, R., Kocks, C., Rajewsky, K., Rajewsky, N., Roes, J.,
Sablitzky, F., Siekevitz, M., Timing, genetic requirements and functional consequences
of somatic hypermutation during B-cell development. Immunol. Rev. 1987.96:5-22
Amsbaugh, DF., Hansen, CT., Prescott, B., Stashak, PW., Barthold, DR., Barker, PJ.,
Genetic control of the antibody response to type 3 pneumococcal polysaccharide in
mice. I. Evidence that an X-linked gene plays a decisive role in determining
responsiveness. J. Exp. Med. 1972.136:931-949
Angelin-Duclos, C., Cattoretti, G., Lin, K-I., Calame, K., Commitment of B
lymphocytes to plasma cell fate is associated with Blimp-1 expression in vivo. J.
Immunol. 2000.165:5462-5471
Arakawa, H., Hauschild, J., Buerstedde, JM., Requirement of the activation-induced
deaminase (AID) gene for immunoglobulin gene conversion. Science. 2002.295:13011306
Arpin, C., Dechanet, J., Van Kooten, C., Merville, P., Grouard, G., Briere, F.,
Banchereau, J., Liu, YJ., Generation of memory B cells and plasma cells in vitro.
Science 1995.268:720-722
Aydar, Y., Balogh, P., Tew, JG., Szakal, AK., Altered regulation of FcgammaRII on
aged follicular dendritic cells correlated with immunoreceptor tyrosine-based inhibition
motif signaling in B cells and reduced germinal center formation. J. Immunol.
2003.171:5975-5987
Azuma, T., Motoyama, N., Fields, LE., Loh, DY., Mutations of the chloroamphenicol
acetyl transferase transgene driven by the immunoglobulin promotor and intron
enhancer. Int. Immunol. 1993.5:121-130
- 40 -
Barberis, A., Widenhorn, K., Vitelli, L., Busslinger, M., A novel B-cell lineage specific
transcription factor present at early but not late stages of differentiation. Genes Dev.
1990.4:849-859
Barrett, DJ., Ayoub, EM., IgG2 subclass restriction of antibody to pneumococcal
polysaccharides. Clin. Exp. Immunol. 1986.63:127-134
Berberich, I., Shu, GL., Clark, EA., Cross-linking CD40 on B cells rapidly activates
nuclear factor-kappa B. J. Immunol. 1994.153:4357-4366
Berek, C., Berger, A., Apel, M., Maturation of the immune response in germinal
centers. Cell. 1991.67:1121-1129
Bernasconi, NL., Traggiai, E., Lanzavecchia, A., Maintenance of serological memory
by polyclonal activation of human memory B cells. Science. 2002.298:2199-2202
Betz, AG., Milstein, C., González-Fernández, A., Pannel, R., Larson, T., Neuberger,
MS., Elements regulating somatic hypermutation of an immunoglobulin kappa gene:
critical role for the intron enhancer/matrix attachment site. Cell. 1994.77:239-248
Betz, AG., Rada, C., Pannel, R., Milstein, C., Neuberger, MS., Passenger transgenes
reveal intrinsic specificity of the antibody hypermutation mechanism: clustering,
polarity, and specific hot spots. Proc. Natl. Acad. Sci. USA. 1993.90:2385-2388
Bouillet, P., Metcalf, D., Huang, DCS., Tarlinton, DM., Kay, TWH., Köntgen, F.,
Adams, JM., Strasser, A., Proapoptotic Bcl-2 relative Bim required for certain apoptotic
responses, leukocyte homeostasis, and to preclude autoimmunity. Science.
1999.286:1735-1738
Bruggemann, MG., Williams, GT., Bindon, CI. Walker, MR., Jefferis, R., Waldmann,
H., Neuberger, MS., Comparison of the effector functions of human immunoglobulins
using a matched set of chimeric antibodies. J. Exp. Med. 1987.166:1351-1361
Buhl, AM, Cambier, JC., Co-receptor and accessory regulation of B-cell antigen
receptor signal transduction. Immunol. Rev. 1997.160:127-138
Burns EA., Lum, LG., Seigneuret, MC., Giddings, BR., Goodwin, JS., Decreased
specific antibody synthesis in old adults: decreased potency of antigen-specific B cells
with aging. Mech. Age. Dev. 1990.53:229-241
Calame, KL., Plasma cells: finding new light at the end of B cell development. Nat.
Immunol. 2001.2:1103-1108
Calame, KL., Lin, K-I., Tunyaplin, C., Regulatory mechanisms that determine the
development and function of plasma cells. Ann. Rev. Immunol. 2003.21:205-230
Carroll, MC., The role of complement and complement receptors in induction and
regulation of immunity. Annu. Rev. Immunol. 1998.16:545-568
- 41 -
Carter, RH., Doody, GM., Bolen, JB., Fearon, DT., Membrane IgM-induced tyrosine
phosphorylation of CD19 requires a CD19 domain that mediates association with
components of the B cell antigen receptor complex. J. Immunol. 1997.158:3062-3069
Carter, RH., Fearon, DT., CD19: Lowering the threshold for antigen receptor
stimulation of B lymphocytes. Science. 1992.256:105-107
Carter, RH., Spycher, MO., Ng, YC., Hoffman, H., Fearon, DT., Synergistic interaction
between complement receptor type 2 and membrane IgM on B lymphocytes. J.
Immunol. 1988.141:457-463
Castle, SC., Clinical relevance of age-related immune dysfunction. Clin. Infect. Dis.
2000.31:578-585
Choe, J., Choi, YS., IL-10 interrupts memory B cell expansion in the germinal center by
inducing differentiation into plasma cells. Eur. J. Immunol. 1998.28:508-818
Choi, MS., Boise, LH., Gottschalk, AR., Quintas, J., Thompson, CB., Klaus, GG., The
role of bcl-XL in CD40-medidated rescue from anti-mu-induced apoptosis in WEHI231 B lymphoma cells. Eur. J. Immunol. 1995.25:1352-1357
Cong, YZ., Rabin, E., Wortis, HH., Treatment of murine CD5-B cells with anti-Ig, but
not LPS, induces surface CD5: two B-cell activation pathways. Int. Immunol.
1991.3:467-476
Coutelier, JP., van der Logt, JT., Heessen, FW., Warnier, G., Van Snick, J., IgG2a
restriction of murine antibodies elicited by viral infections. J. Exp. Med. 1987.165:64-69
Coutinho, A., Möller, G., Editorial: Immune activation of B cells: evidence for ‘one
nonspecific triggering signal’ not delivered by the Ig receptors. Scand. J. Immunol.
1974.3:133-146
Coutinho, A., Möller, G., Thymus-independent B cell induction and paralysis. Adv.
Immunol. 1975.21:113-236
Coutinho, A., Möller, G., Richter, W., Molecular basis of B cell activation. I.
Mitogenicity of native and substituted dextrans. Scand. J. Immunol. 1974.3:321-328
Craig, SW., Cebra, JJ., Peyer’s patches: an enriched source of precursors for IgAproducing immunocytes in the rabbit. J. Exp. Med. 1971.134:188-200
Croix, D., Ahearn, JM., Rosengard, AM., Han, S., Kelsoe, G., Ma, M., Carroll, MC.,
Antibody response to a T-dependent antigen requires B cell expression of complement
receptors. J. Exp. Med. 1996.183:1857-1864
Daëron, M., Fc receptor biology. Annu. Rev. Immunol. 1997.15:203-234
- 42 -
Dangl, JL., Wensel, TG., Morrison, SL., Stryer, L., Herzenberg, LA., Oi, VT.,
Segmental flexibility and complement fixation of genetically engineered chimeric
human, rabbit and mouse antibodies. EMBO J. 1988.7:1989-1994
Dent, AL., Shaffer, AL., Yu, X., Allman, D., Staudt, LM., Control of inflammation,
cytokine expression, and germinal center formation by BCL-6. Science. 1997.276:589592
de Vinuesa, CG., Cook, MC., Ball, J., Drew, M., Sunners, Y., Cascalho, M., Wabl, M.,
Klaus, CG., MacLennan, IC:, Germinal centers without T cells. J. Exp. Med.
2000.191:485-494
Diaz, M., Casali, P., Somatic immunoglobulin hypermutation. Curr. Opin. Immunol.
2002.14:235-240
Dintzis, HM., Dintzis, RZ., Vogelstein, B., Molecular determinants of immunogenicity:
The immunon model of immune response. Proc. Natl. Acad. Sci. USA. 1976.73:36713675
Do, RK., Hatada, E., Lee, H., Tourigny, MR., Hilbert, D., Chen-Kiang, S., Attenuation
of apoptosis underlies B lymphocyte stimulatory enhancement of humoral immune
response. J. Exp. Med. 2000.192:953-964
Dolmetsch, RE., Lewis, RS., Goodnow, CC., Healy, JI., Differential activation of
transcription factors induced by Ca2+ response amplitude and duration. Nature.
1997.386:855-858
Doria, G., D´Agostaro, G., Poretti, A., Age dependent variations of antibody avidity.
Immunology. 1978.35:601-611
Dörner, T., Brezinschek, RI., Foster, SJ., Domiati, R., Lipsky, PE., Analysis of the
frequency and pattern of somatic mutations within nonproductively rearranged human
variable heavy chain genes. J. Immunol. 1997.158:2779-2789
Driver, DJ., McHeyzer-Williams, LJ., Cool, M., Stetson, DB., McHeyzer-Williams,
MG., Development and maintenance of a B220- memory B cell compartment. J.
Immunol. 2001.167:1393-1405
Dullforce, P., Sutton, DC., Heath, AW., Enhancement of T cell-independent immune
responses in vivo by CD40 antibodies. Nat. Med. 1998.4:88-91
Enders, A., Bouillet, P., Puthalakath, H., Xu, Y., Tarlinton, DM., Strasser, A., Loss of
the pro-apoptotic BH3-only Bcl-2 family member Bim inhibits BCR stimulationinduced apoptosis and deletion of autoreactive B cells. J. Exp. Med. 2003.198:11191126
- 43 -
Endres, RO., Kuchnir, E., Kappler, JW., Marrack, P., Kinsky, SC., A requirement for
nonspecific T cell factors in antibody responses to “T cell independent” antigens. J.
Immunol. 1983.130:781-789
Engel, P., Zhou, LJ., Ord, DC., Sato, S., Koller, B., Tedder, TF., Abnormal B
lymphocyte development, activation, and differentiation in mice that lack or
overexpress the CD19 signal transduction molecule. Immunity. 1995.3:39-50
Ey, PL., Russel-Jones, GJ., Jenkins, CR., Isotypes of mouse IgG-I. Evidence for “noncomplement-fixing” IgG1 antibodies and characterization of their capacity to interfere
with IgG2 sensitization of target red blood cells for lysis by complement. Mol. Immunol.
1980.17:699-710
Fagarasan, S., Kinoshita, K., Muramatsu, M., Ikuta, K., Honjo, T., In situ class
switching and differentiation to IgA-producing cells in the gut lamina propria. Nature.
2001.413:639-643
Falini, B., Fizzotti, M., Pucciarini, A., Bigerna, B., Marafioti, T., Gambacorta, M.,
Pacini, R., Alunni, C., Natali-Tanci, L., Ugolini, B., Sebastiani, C., Cattoretti, G., Pileri,
S., Dalla-Favera, R., Stein, H., A monoclonal antibody (MUMIp) detects expression of
the MUMI/IRF4 protein in a subset of germinal center B cells, plasma cells, and
activated T cells. Blood. 2000.95:2084-2092
Fang, W., Weintraub, BC., Dunlap, B., Garside, P., Pape, KA., Jenkins, MK.,
Goodnow, CC., Mueller, DL., Behrens, TW., Self-reactive B lymphocytes
overexpressing Bcl-xL escape negative selection and are tolerized by clonal anergy and
receptor editing. Immunity. 1998.9:35-45
Fernández, C., Genetic mechanisms for dominant VH gene expression. The VHB512
gene. J. Immunol. 1992.149:2328-36
Fernández, C., Lieberman, R., Möller, G., The immune response to the alpha 1-6
epitope of dextran is determined by a gene linked to the IgCH locus. Scand. J. Immunol.
1979.10:77-80
Fernández, C., Möller, G., Immunological unresponsiveness to thymus-independent
antigens. Two fundamentally different genetic mechanisms of B-cell unresponsiveness
to dextran. J. Exp. Med. 1977.146:1663-1677
Fisher, CL., Ghysdael, J., Cambier, JC., Ligation of membrane Ig leads to calciummediated phosphorylation of the proto-oncogene product Est-1. J. Immunol.
1991.146:1743-1749
Fliedner, TM., Kesse, M., Crokite, EP., Robertson, JS., Cell proliferation in germinal
centers of the rat spleen. Ann. N.Y. Acad. Sci. 1964.113:578-594
Foote, J., Milstein, C., Kinetic maturation of an immune response. Nature.
1991.352:530-532
- 44 -
Foy, TM., Laman, JD., Ledbetter, JA., Aruffo, A., Claassen, E., Noelle, RJ., gp39CD40 interactions are essential for germinal center formation and the development of B
cell memory. J. Exp. Med. 1994.180:157-163
Fuller, KA., Kanagawa, O., Nahm, MH., T cells within germinal centerns are specific
for the immunizing antigen. J. Immunol. 1993.151:4501-4512
Garcia de Vinuesa, C., MacLennan, ICM., Holman, M., Klaus, GG., Anti-CD40
antibody enhances responses to polysaccharide without mimicking T cell help. Eur. J.
Immunol. 1999.29:3216-3224
Garrone, P., Neidhardt, EM., Garcia, E., Galibert, L., van Kooten, KC., Banchereau, J.,
Fas ligation induces apoptosis of CD40-activated human B lymphocytes. J. Exp. Med.
1995.182:1265-1273
Gay, D., Saunders, T., Camper, S., Weigert, M., Receptor editing: an approach by
autoreactive B cells to escape tolerance. J. Exp. Med. 1993.177:999-1008
Goidl, EA., Innes, JB., Weksler, ME., Immunological studies of aging. II. Loss of IgG
and high avidity plaque forming cells and increased suppressor cell activity in aging
mice. J. Exp. Med. 1976.144:1037-1048
Gold, MR., To make antibodies or not: signaling by the B-cell antigen receptor. Trends.
Pharmacol. Sci. 2002.23:316-324
Gorski, J., Rollini, P., Mach, B., Somatic mutations of immunoglobulin variable genes
are restricted to the rearranged V gene. Science. 1983.220:1179-1181
Gray, D., Skarvall, H., B-cell memory is short-lived in the absence of antigen. Nature.
1988.336:70-73
Gross, JA., Dillon, SR., Mudri, S., Johnston, J., Littau, A., Roque, R., Rixon, M.,
Schou, O., Foley, KP., Haugen, H., McMillen, S., Waggie, K., Schreckhise, RW.,
Shoemaker, K., Vu, T., Moore, M., Grossman, A., Clegg, CH., TACI-Ig neutralizes
molecules critical for B cell development and autoimmune disease: impaired B cell
maturation in mice lacking BLys. Immunity. 2001.15:289-302
Gross, JA., Johnston, J., Mudri, S., Enselman, R., Dillon, SR., Madden, K., Xu, W.,
Parrish-Novak, J., Foster, D., Lofton-Day. C., Moore, M., Littau, A., Grossman, A.,
Haugen, H., Foley, K., Blumberg, H., Harrison, K., Kindsvogel, W., Clegg, CH., TACI
and BCMA are receptors for a TNF homologue implicated in B cell autoimmune
disease. Nature. 2000.404:995-999
Haas, KM., Estes, DM., Activation of bovine B cells via surface immunoglobulin M
cross-linking or CD40 ligation results in different B-cell phenotypes. Immunology.
2000.99:272-278
- 45 -
Hackett, J Jr., Rogerson, BJ., O’Brien, RL., Storb, U., Analysis of somatic mutations in
kappa transgenes. J. Exp. Med. 1990.172:131-137
Hardy, RR., Carmack, CE., Shinton, SA., Kemp, JD., Hayakawa, K., Resolution and
characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J.
Exp. Med. 1991.173:1213-1225
Harris, RS., Sale, JE., Petersen-Mahrt, SK., Neuberger, MS., AID is essential for
immunoglobulin V gene conversion in a cultured B cell line. Curr. Biol. 2002.12:435438
Hartley, SB., Cooke, MP., Fulcher, DA., Harris, AW., Cory, S., Basten, A., Goodnow,
CC., Elimination of self-reactive B lymphocytes proceeds in two stages: arrested
development and cell death. Cell. 1993.72:325-335
Hippen, KL., Buhl, AM., D’Ambrosio, D., Nakamura, K., Persin, C., Cambier, JC.,
FcRγIIB1 inhibition of BCR-mediated phophoinositide hydrolysis and Ca2+
mobilization is integrated by CD19 dephosphorylation. Immunity. 1997.7:49-58
Honjo, T., Kinoshita, K., Muramatsu, M., Molecular mechanism of class switch
recombination: linkage with somatic hypermutation. Annu. Rev. Immunol. 2002.20:165196
Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K.,
Akira, S., Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive
to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol.
1999.162:3749-3752
Hosokawa, T., Studies on B-cell memory. II. T-cell independent antigen can induce Bcell memory. Immunology. 1979.38:291-299
Hosokawa, T., Aoike, A., Hosono, M., Motoi, S., Kawai, K., Studies on B-cell memory.
IV. Effects of lipopolysaccharide on primary and secondary responses to T-independent
type-2 (TI-2) antigen in mice. Microbiol. Immunol. 1989.33:941-949
Hsu, BL., Harless, SM., Lindsley, RC., Hilbert, DM., Cancro, MP., Cutting edge: BLys
enables survival of transitional and mature B cells through distinct mediators. J.
Immunol. 2002.168:5993-5996
Ivars, F., Nyberg, G., Holmberg, D., Coutinho, A., Immune response to bacterial
dextrans. II. T cell control of antibody isotypes. J. Exp. Med. 1983.158:1498-1510
Jacob, J., Kassir, R., Kelsoe, G., In situ studies of the primary immune response to (4hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell
populations. J. Exp. Med. 1991a.173:1165-1175
Jacob, J., Kelso, G., Rajewsky, K., Weiss, U., Intraclonal generation of antibody
mutants in germinal centers. Nature 1991b.354:389-392
- 46 -
Kaisho, T., Schwenk, F., Rajewsky, K., The roles of gamma 1 heavy chain membrane
expression and cytoplasmatic tail in IgG1 responses. Science 1997.276:412-415
Karasuyama, H., Rolink, A., Melchers, F., Surrogate light chain in B cell development.
Adv. Immunol. 1996.63:1-41
Kawabe, T., Naka, T., Yoshida, K., Tanaka, T., Fujiwara, H., Suematsu, S., Yoshida,
N., Kishimoto, T., Kikutani, H., The immune responses in CD40-deficient mice:
impaired immunoglobulin class switching and germinal center formation. Immunity.
1994.1:167-178
Kayagaki, N., Yan, M., Seshasayee, D., Wang, H., Lee, W., French, DM., Grewal, IS.,
Cochran, AG., Gordon, NC., Yin, J., Starovasnik, MA., Dixit, VM., BAFF/BLyS
receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface loop
and promotes processing of NF-κB2. Immunity. 2002.17:515-524
Kelsoe, G., In situ studies of the germinal center reaction. Adv. Immunol. 1995.60:267288
Kelsoe, G., Zheng, B., Sites of B-cell activation in vivo. Curr. Opin. Immunol.
1993.5:418-422
Kenter, AL., Class-switch recombination: after the dawn of AID. Curr. Opin. Immunol.
2003.15:190-198
Khare, SD., Sarosi, I., Xia, XZ., McCabe, S., Miner, K., Solovyev, I., Hawkins, N.,
Kelly, M., Chang, D., Van G., Ross, L., Delaney, J., Wang, L., Lacey, D., Boyle, WJ.,
Hsu, H., Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice.
Proc. Natl. Acad. Sci. USA. 2000.97:3370-3375
Kinoshita, K., Honjo, T., Linking class-switch recombination with somatic
hypermutation. Nat. Rev. Mol. Cell. Biol. 2001.2:493-503
Kishimoto, S., Takahama, T., Mizumachi, M., In vitro immune responses to the 2,4,6trinitrophenyl determinant in aged C57BL/6 mice: changes in the humoral response to,
avidity for the TNP determinant, and responsiveness to LPS effect with aging. J.
Immunol. 1976.116:294-300
Klein, U., Kuppers, R., Rajewsky, K., Evidence for a large compartment of IgMexpression memory B cells in humans. Blood. 1997.89:1288-1298
Knight, JA., The process and theories of aging. Ann. Clin. Lab. Sci. 1995.25:1-12
Kolb, C., Fuchs, B., Weiler, E., The thymus-independent antigen alpha(1-3) dextran
elicits proliferation of precursors for specific IgM antibody-producing cells (memory
cells), which are revealed by LPS stimulation in soft agar cultures and detected by
immunoblot. Eur. J. Immunol. 1993.23:2959-2966
- 47 -
Koopman, G., Keehnen, RM., Lindhout, E., Zhou, DF., de Groot, C., Pals, ST.,
Germinal center B cells rescued from apoptosis by CD40 ligation or attachment to
follicular dendritic cells, but not by engagement of surface immunoglobulin or adhesion
receptors, become resistant to CD95-induced apoptosis. Eur. J. Immunol. 1997.27:1-7
Kosco-Vilbois, MH., Zentgraf, H., Gerdes, J., Bonnefoy, JY., To’B’ or not to ‘B’ a
germinal center? Immunol. Today. 1997.18:225-230
Kurosaki, T., Maeda, A., Ishiai, M., Hashimoto, A., Inabe, K., Takata, M., Regulation
of the phospholipase C-γ2 pathway in B cells. Immunol. Rev. 2000.176:19-29
Kuss, AW., Knödel, M., Berberich-Siebelt, F., Lindermann, D., Schimpl, A., Berberich,
I., A1 expression is stimulated by CD40 in B cells and rescues WEHI 231 cells from
anti-IgM-induced cell death. Eur. J. Immunol. 1999.29:3077-3088
Kutteh, WH., Prince, SJ., Mestecky, J., Tissue origins of human polymeric and
monomeric IgA. J. Immunol. 1982.128:990-995
Laâbi, Y., Egle, A., Strasser, A., TNF cytokine family: more BAFF-ling complexities.
Curr. Biol. 2001.11:R1013-1016
Lam, KP., Kuhn, R., Rajewsky, K., In vivo ablation of surface immunoglobulin on
mature B cells by inducible gene targeting results in rapid cell death. Cell.
1997.90:1073-1083
Lang, J., Arnold, B., Hammerling, G., Harris, AW., Korsmeyer, S., Russell, D.,
Strasser, A., Nemazee, D., Enforced Bcl-2 expression inhibits antigen-mediated clonal
elimination of peripheral B cells in an antigen dose-dependent manner and promotes
receptor editing in autoreactive, immature B cells. J. Exp. Med. 1997.186:1513-1522
Lebecque, SG., Gearhart, PJ., Boundaries of somatic mutation in rearranged
immunoglobulin genes: 5' boundary is near the promoter, and 3' boundary is
approximately 1 kb from V(D)J gene. J. Exp. Med. 1990.172:1717-1727
Letvin, NL., Benacerraf, B., Germain, RN., B lymphocyte responses to trinitrophenylconjugated Ficoll: requirement for T lymphocyte and Ia bearing adherent cells. Proc.
Natl. Acad. Sci. USA. 1981.78:5113-5117
Lin, KI., Angelin-Duclos, C., Kuo, TC., Calame, K., Blimp-1 dependent repression of
Pax-5 is required for differentiation of B cells to immunoglobulin M-secreting plasma
cells. Mol. Cell. Biol. 2002.22:4771-4780
Lin, Y., Wong, K., Calame, K., Repression of c-myc transcription by Blimp-1, an
inducer of terminal B cell differentiation. Science. 1997.276:596-599
Liu, AH., Jena, PK., Wysocki, LJ., Tracking the development of single memory-lineage
B cells in a highly defined immune response. J. Exp. Med. 1996.183:2053-2063
- 48 -
Liu, YJ., Sites of B lymphocyte selection, activation, and tolerance in spleen. J. Exp.
Med. 1997.186:625-629
Liu, YJ., Oldfield, S., MacLennan, ICM., Memory B cells in T cell-dependent antibody
responses colonize the splenic marginal zones. Eur. J. Immunol. 1988.18:355-362
Liu, YJ., Zhang, J., Lane, PJ., Chan, EY., MacLennan, ICM., Sites of specific B cell
activation in primary and secondary responses to T cell-dependent and T cellindependent antigens. Eur. J. Immunol. 1991.21:2951-2962
Mackay, F., Woodcock, SA., Lawton, P., Ambrose, C., Baetscher, M., Schneider, P.,
Tschopp, J., Browning, JL., Mice transgenic for BAFF develop lymphocytic disorders
along with autoimmune manifestations. J. Exp. Med. 1999.190:1697-1710
MacLennan, IC., Gray, D., Antigen-driven selection of virgin and memory B cells.
Immunol. Rev. 1986.91:61-85
MacLennan, IC., Johnson, GD., Liu, YJ., Gordon, J., The heterogeneity of follicular
reactions. Res. Immunol. 1991.142:253-257
MacLennan, IC., Liu, YJ., Johnson, GD., Maturation and dispersal of B-cell clones
during T cell-dependent antibody responses. Immunol. Rev. 1992.126:143-161
MacLennan, IC., Toellner, KM., Cunningham, AF., Serre, K., Sze, DM., Zuniga, E.,
Cook, MC., Vinuesa, CG., Extrafollicular antibody responses. Immunol. Rev.
2003.194:8-18
Macpherson, AJ., Gatto, D., Sainsbury, E., Harriman, GR., Hengartner, H., Zinkernagel,
RM., A primitive T cell-independent mechanism of intestinal mucosal IgA responses to
commensal bacterial. Science. 2000.288:2222-2226
Manz, RA., Thiel, A., Radbruch, A., Lifetime of plasma cells in the bone marrow.
Nature. 1997.388:133-134
Mariuzza, RA., Poljak, RJ., Schwarz, FP., The energetics of antigen-antibody binding.
Res. Immunol. 1994.145:70-72
Marsters, SA., Yan, M., Pitti, RM., Haas, PE., Dixit, VM., Ashkenazi, A., Interaction of
the TNF homologues BLyS and APRIL with the TNF receptor homologues BCMA and
TACI. Curr. Biol. 2000.10:785-788
Martin, SW., Goodnow, CC., Burst-enhancing role of the IgG membrane tail as a
molecular determinant of memory. Nat. Immunol. 2002.3:182-188
Maruyama, M., Lam, KP., Rajewsky, K., Memory B-cell persistence is independent of
persisting immunizing antigen. Nature. 2000.407:636-642
- 49 -
Matsumoto, AK., Kopicky-Burd, J., Carter, RH., Tuveson, DA., Tedder, TF., Fearon,
DT., Intersection of the complement and immune systems: A signal transduction
complex of the B lymphocyte-containing complement receptor type 2 and CD19. J.
Exp. Med. 1991.173:55-64
Mazel, S., Burtrum, D., Petrie, HT., Regulation of cell division cycle progression by
bcl-2 expression: a potential mechanism for inhibition of programmed cell death. J.
Exp. Med. 1996.183:2219-2226
McHeyzer-Williams, LJ., Cool, M., McHeyzer-Williams, MG., Antigen-specific B cell
memory expression and replenishment of a novel B220- memory B cell compartment. J.
Exp. Med. 2000.191:1149-1165
McHeyzer-Williams, MG., McLean, MJ., Lalor, PA., Nossal, GJ., Antigen-driven B cell
differentiation in vivo. J. Exp. Med. 1993.178:295-307
McKean, D., Huppi, K., Bell, M., Staudt, L., Gerhard, W., Weigert, M., Generation of
antibody diversity in the immune response of BALB/c mice to influenza virus
hemagglutinin. Proc. Natl. Acad. Sci. USA. 1984.81:3180-3184
Meyer, KB., Sharpe, MJ., Surani, MA., Neuberger, MS., The importance of the 3'enhancer region in immunoglobulin kappa gene expression. Nucleic. Acids. Res.
1990.18:5609-5615
Michaelson, JS., Singh, M., Birshtein, BK., B cell lineage-specific activator protein
(BSAP). A player at multiple stages of B cell development. J. Immunol. 1996.156:23492351
Miller, C., Kelsoe, G., Ig VH hypermutation is absent in the germinal centers of aged
mice. J. Immunol. 1995.155:3377-3384
Miller, RA., The aging immune system: primer and prospectus. Science. 1996.273:7074
Miller, RA., Aging and immune function. Int. Rev. Cytol. 1991.124:187-215
Mittrücker, HW., Matsuyama, T., Grossman, A., Kunding, TM., Potter, J., Shahinian,
A., Wakeham, A., Patterson, B., Ohashi, PS., Mak, TW., Requirement of the
transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science.
1997.275:540-543
Molina, H., Holers, VM., Li, B., Fung, Y., Mariathasan, S., Goellner, J., StraussSchoenberger, J., Karr, RW., Chaplin, DD., Markedly impaired humoral immune
response in mice deficient in complement receptors 1 and 2. Proc. Natl. Acad. Sci. USA.
1996.93:3357-3361
- 50 -
Möller, G., Receptors for innate pathogen defence in insects are normal activation
receptors for specific immune responses in mammals. Scand. J. Immunol. 1999.50:341347
Möller, G., Fernandez, C., Alarcon-Riquelme, M., Thymus-independent antigens. In
Snow, EC., (Eds.) T-cell dependent and independent B-cell activation. CRC Press, Boca
Raton 1991, pp 65-74
Mond, JJ., Lees, A., Snapper, C., T cell-independent antigens type 2. Annu. Rev.
Immunol. 1995.13:655-692.
Mond, JJ., Mongini, PKA., Sieckmann, D., Paul, WE., Role of T lymphocytes in
response to TNP-AECM-Ficoll. J. Immunol. 1980.125:1066-1070
Moore, PA., Belvedere, O., Orr, A., Pieri, K., LaFleur, DW., Feng, P., Soppet, D.,
Charters, M., Gentz, R., Parmelee, D., Li, Y., Galperina, O., Giri, J., Roschke, V.,
Nardelli, B., Carrell, J., Sosnovtseva, S., Greenfield, W., Ruben, SM., Olsen, HS.,
Fikes, J,. Hilbert, DM., BLyS:member of the tumor necrosis factor family and B
lymphocyte stimulator. Science. 1999.285:260-263
Morrison, AM., Nutt, SL., Thevenin, C., Rolink, A., Busslinger, M., Loss- and gain-offunction mutations reveal an important role of BSAP (Pax5) at the start and end of B
cell differentiation. Semin. Immunol. 1998.10:133-142
Motoyama, N., Miwa, T., Suzuki, Y., Okada, H., Azuma, T., Comparison of somatic
mutation frequency among immunoglobulin genes. J. Exp. Med. 1994.179:395-403
Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., Honjo, T., Class
switch recombination and hypermutation require activation-induced cytidine deaminase
(AID), a potential RNA editing enzyme. Cell. 2000.102:553-563
Nemazee, D., Receptor selection in B and T lymphocytes. Annu. Rev. Immunol.
2000.18:19-51
Neuberger, MS., Rajewsky, K., Activation of mouse complement by monoclonal
antibodies. Eur. J. Immunol. 1981.11:1012-1016
Nicoletti, C., Borghesi-Nicoletti, C., Yang, X., Schulze, DH., Cerny, J., Repertoire
diversity of antibody response to bacterial antigens in aged mice. II. Phosphorylcholine
antibody in young and aged mice differ in both VH/VL gene repertoire and in
specificity. J. Immunol. 1991.147:2750-2755
Nieuwenhuis, P., Ford, WL., comparative migration of B- and T-lymphocytes in the rat
spleen and lymph nodes. Cell. Immunol. 1976.23:254-267
Nieuwenhuis, P., Opstelten, D., Functional anatomy of germinal centers. Am. J. Anat.
1984.170:421-435
- 51 -
Niiro, H., Clark, EA., Regulation of B-cell fate by antigen-receptor signals. Nat. Rev.
Immunol. 2002.2:945-956
Nisitani, S., Tsubata, T., Murakami, M., Okamoto, M., Honjo, T., The bcl-2 gene
product inhibits clonal deletion of self-reactive B lymphocytes in the periphery but not
in the bone marrow. J. Exp. Med. 1993.178:1247-1254
Niu, H., Ye, BH., Dalla-Favera, R., Antigen receptor signaling induces MAP kinasemediated phosphorylation and degradation of the BCL-6 transcription factor. Genes.
Dev. 1998.12:1953-1961
Oldfield, S., Liu, YJ., Beaman, M., MacLennan, CM., Memory B cells generated in T
cell-dependent antibody responses colonise the splenic marginal zone. Adv. Exp. Med.
Biol. 1988.237:93-98
O’Reilly, L., Huang, DCS., Strasser, A., The cell death inhibitor Bcl-2 and its
homologues influence control of cell cycle entry. EMBO J. 1996.15:6979-90
Osmond, DG., The turnover of B-cell populations. Immunol. Today. 1993.14:34-37
Osmond, DG., Rolink, A., Melchers, F., Murine B lymphopoiesis: towards a unified
model. Immunol. Today. 1998.19:65-68
Palmer, E., Negative selection – clearing out the bad apples from the T-cell repertoire.
Nat. Rev. Immunol. 2003.3:383-391
Papadea, C., Check, IJ., Human IGG and IGG subclasses: biochemical, genetic, and
clinical aspects. Crit. Rev. Clin. Lab. Sci. 1989.27:27-58
Perlmutter, R., Hansburg, D., Briles, DE., Nicolotti, R., Davie, JM., Subclass restriction
of murine anti-carbohydrate antibodies. J. Immunol. 1978.121:566-572
Peters, A., Storb, U., Somatic hypermutation of immunoglobulin genes is linked to
transcription initiation. Immunity 1996.4:57-65
Petersen-Mahrt, SK., Harris, RS., Neuberger, MS., AID mutates E.coli suggesting a
DNA deamination mechanism for antibody diversification. Nature. 2002.418:99-103
Phillips, NE., Parker, DC., Cross-linking of B lymphocyte Fc gamma receptors and
membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis. J.
Immunol. 1984.132:627-632
Phillips, NE., Parker, DC., Subclass specificity of Fcg receptor-mediated inhibition of
mouse B cell activation. J. Immunol. 1985.134:2835-2838
Piskurich, JF., Lin, KI., Lin, Y., Wang, Y., Ting, JP., Calame, K., BLIMP-1 mediates
extinction of major histocompatibility class II transactivator expression in plasma cells.
Nat. Immunol. 2000.1:526-532
- 52 -
Poltorak, A:, He, X., Smirnova, I., Liu, MY., Van Huffel, C., Du, X., Birdwell, D.,
Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton,
B., Beutler, B., Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice:
mutations in Tlr4 gene. Science. 1998.282:2085-2088
Radic, MZ., Erikson, J., Litwin, S., Weigert, M., B lymphocytes may escape tolerance
by revising their antigen receptors. J. Exp. Med. 1993.177:1165-1173
Rathmell, JC., Goodnow, CC., Effects of the lpr mutation on elimination and
inactivation of self-reactive B cells. J. Immunol. 1994.153:2831-2842
Ravetch, JV., Kinet, JP., Fc receptors. Annu. Rev. Immunol. 1991.9:457-492
Reimold, AM., Iwakoshi, NN., Manis, J., Vallabhajosyula, P., Szomolanyi-Tsuda, E.,
Gravallese, EM., Friend, D., Grusby, MJ., Alt, F., Glimcher, LH., Plasma cell
differentiation requires the transcription factor XBP-1. Nature. 2001.412:300-307
Reimold, AM., Ponath, PD., Li, YS., Hardy, RR., David, CS., Strominger, JL.,
Glimcher, LH., Transcription factor B cell lineage-specific activator protein regulates
the gene for human X-box binding protein 1. J. Exp. Med. 1996.183:393-401
Reth, M., Wienands, J., Initiation and processing of signals from the B cell antigen
receptor. Annu. Rev. Immunol. 1997.15:453-479
Revy, P., Muto, T., Levy, Y., Geissmann, F., Plebani, A., Sanal, O., Catalan, N.,
Forveille, M., Dufourcq-Labelouse, R., Gennery, A., Tezcan, I., Ersoy, F., Kayserili, H.,
Ugazio, AG., Brousse, N., Muramatsu, M., Notarangelo, LD., Kinoshita, K., Honjo, T.,
Fischer, A., Durandy, A., Activation-induced cytidine deaminase (AID) deficiency
causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell.
2000.102:565-575
Reynaud, CA., García, C., Hein, WR., Weill, J-C., Hypermutation generating the sheep
immunoglobulin repertoire is an antigen-independent process. Cell. 1995.80:115-125
Rickert, RC., Rajewsky, K., Roes, J., Impairment of T-cell-dependent B-cell responses
and B-1 cell development in CD19-deficient mice. Nature. 1995.376:352-355
Riesen, WF., Skvaril, F., Braun, DG., Natural infection of man with group A
streptococci. Levels; restriction in class, subclass, and type; and clonal appearance of
polysaccharide group-specific antibodies. Scand. J. Immunol. 1976.5:383-390
Rinkenberger, JL., Wallin, JJ., Johnson, KW., Koshland, ME., An interleukin-2 signal
relieves BSAP (Pax5)-mediated repression of the immunoglobulin J chain gene.
Immunity. 1996.5:377-386
Roes, J., Hüppi, K., Rajewsky, K., Sablitzky, F., V gene rearrangement is required to
fully activate the hypermutation mechanism in B cells. J. Immunol. 1989.142:10221026
- 53 -
Rose, ML., Birbeck, MS., Wallis, VJ., Forrester, JA., Davies, AJ., Peanut lectin binding
properties of germinal centers of mouse lymphoid tissue. Nature. 1980.284:364-366
′
Rothenfluh, HS., Taylor, L., Bothwell, ALM., Both, GW., Steele, EJ., Somatic
hypermutation in 5 flanking regions of heavy chain antibody variable regions. Eur. J.
Immunol. 1993.23:2152-2159
Rothstein, TL., Wang, JKM., Panka, DJ., Foote, LC., Wang, Z., Stanger, B., Cui, H., Ju,
S-T., Marshak-Rothstein, A., Protection against Fas-dependent Th1-mediated apoptosis
by antigen receptor engagement in B cells. Nature. 1995.374:163-165
Rubio, CF., Kench, J., Russell, DM., Yawger, R., Nemazee, D., Analysis of central B
cell tolerance in autoimmune-prone MRL/lpr mice bearing autoantibody transgenes. J.
Immunol. 1996.157:65-71
Schattner, EJ., Elkon, KB., Yoo, DH., Tumang, J., Krammer, PH., Crow, MK.,
Friedman, SM., CD40 ligation induces Apo-1/Fas expression on human B lymphocytes
and facilitates apoptosis through the Apo-1/Fas pathway. J. Exp. Med. 1995.182:15571565
Scher, I., The CBA/N mouse strain: an experimental model illustrating the influence of
the X-chromosome on immunity. Adv. Immunol. 1982.33:1-71
Schiemann, B., Gommerman, JL., Vora, K., Cachero, TG., Shulga-Morskaya, S.,
Dobles, M., Frew, E., Scott, ML., An essential role for BAFF in the normal
development of B cells through a BCMA-independent pathway. Science.
2001.293:2111-2114
Schliephake, DE., Schimpl, A., Blimp-1 overcomes the block in IgM secretion in
lipopolysaccharide/anti- µ F(ab )2-co-stimulated B lymphocytes. Eur. J. Immunol.
1996.26:568-271
′
Schneider, P., MacKay, F., Steiner, V., Hofmann, K., Bodmer, J-L., Holler, N.,
Ambrose, C., Lawton, P., Bixler, S., Acha-Orbea, H., Valmori, D., Romero, P., WernerFavre, C., Zubler, RH., Browning, JL., Tschopp, J., BAFF, a novel ligand of the tumor
necrosis factor family, stimulates B cell growth. J. Exp. Med. 1999.189:1747-1756
Schneider, P., Tschopp, J., BAFF and the regulation of B cell survival. Immunol. Lett.
2003.88:57-62
Schwarz, YX., Yang, M., Qin, D., Wu, J., Jarvis, WD., Grant, S., Burton, GF., Szakal,
AK., Tew, JG., Follicular dendritic cells protect malignant B cells from apoptosis
induced by anti-Fas and antineoplastic agents. J. Immunol. 1999.163:6442-6447
Scott, MG., Fleischman, JB., Preferential idiotype-isotype association in antibodies to
dinitrophenyl antigens. J. Immunol. 1982.128:2622-2628
- 54 -
Shaffer, AL., Yu, X., He, Y., Boldrick, J., Chan, EP., Staudt, LM., BCL-6 represses
genes that function in lymphocyte differentiation, inflammation, and cell cycle control.
Immunity. 2000.13:199-212
Shapiro-Shelef, M., Lin, KI., McHeyzer-Williams, LJ., Liao, J., McHeyzer-Williams,
MG., Calame, K., Blimp-1 is required for the formation of immunoglobulin secreting
plasma cells and pre-plasma memory B cells. Immunity. 2003.19:607-620
Shlomchik, MJ., Aucoin, AH., Pisetsky, DS., Weigert, MG., Structure and function of
anti-DNA autoantibodies derived from a single autoimmune mouse. Proc. Natl. Acad.
Sci. USA. 1987.84:9150-9154
Siegrist, CA., Neonatal and early life vaccinology. Vaccine. 2001.19:3331-3346
Singh, M., Birshtein, BK., NF-HB (BSAP) is a repressor of the murine immunoglobulin
heavy-chain 3 α enhancer at early stages of B-cell differentiation. Mol. Cell.
Biol.1993.13:3611-3622
′
Skvaril, F., Schilt, U., Characterization of the subclass and light chain types of IgG
antibodies to rubella. Clin. Exp. Immunol. 1984.55:671-676
Slack, JH., Der-Balian, G., Nahmn, MH., Davie, JM., Subclass restriction of murine
antibodies. II. The IgG plaque-forming response to thymus-independent type 1 and type
2 antigen in normal mice and mice expressing an X-linked immunodeficiency. J. Exp.
Med. 1980.151:853-862
Slifka, MK., Antia, R., Whitmite, JK., Ahmed, R., Humoral immunity due to long-loved
plasma cells. Immunity. 1998.8:363-372
Smith, DS., Creadon, G., Jena, PK., Portanova, JP., Kotzin, BL., Wysocki, LI., Di- and
trinucleotide target preferences of somatic mutagenesis in normal and autoreactive B
cells. J. Immunol. 1996.156:2642-2652
Smith, KGC., Light, A., Nossal, GJV., Tarlington, DM., The extent of affinity
maturation differs between memory and antibody-forming cell compartments in the
primary immune response. EMBO J. 1997.16:2996-3006
Smith, KGC., Light, A., O´Reilly, L., Ang, S-M., Strasser, A., Tarlinton, D., bcl-2
transgene expression inhibits apoptosis in the germinal center and reveals differences in
the selection of memory B cells and bone marrow antibody forming cells. J. Exp. Med.
2000.191:475-484
Smith, KG., Nossal, GJ., Tarlinton, DM., FAS is highly expressed in the germinal
center but is not required for regulation of the B-cell response to antigen. Proc. Natl.
Acad. Sci. USA. 1995.92:11628-11632
- 55 -
Smith, KGC., Weiss, U., Rajewsky, K., Nossal, GJV., Tarlinton, DM., Bcl-2 increases
memory B cell recruitment but does not perturb selection in germinal centers. Immunity.
1994.1:808-813
Smith, AM., The affect of age on the immune response to type III pneumococcal
polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) in BALB/c, SJL/J, and
C3H mice. J. Immunol. 1976.166:469-474
Snapper, CM., Kehry, MR., Castle, BR., Mond, JJ., Multivalent, but not divalent,
antigen receptor cross-linkers synergize with CD40 ligand for induction of Ig synthesis
and class switching in normal murine B cells. A redefinition of the TI-2 versus T celldependent antigen dichotomy. J. Immunol. 1995.154:1177-1187
Snapper, CM., Marcu, KB., Zelazowski, P., The immunoglobulin class switch: beyond
”accessibility”. Immunity. 1997.6:217-223
Snapper, CM., Mond, JJ., A model for induction of T cell-independent humoral
immunity in response to polysaccharide antigen. J. Immunol. 1996.157:2229-2233
Song, H., Price, PW., Cerny, J., Age-related changes in antibody repertoire: contribution
from T cells. Immunol. Rev. 1997.160:55-62
Stavnezer, J., Immunoglobulin class switching. Curr. Opin. Immunol. 1996.8:199-205
Stein, JV., Lopez-Fraga, M., Elustondo, FA., Carvalho-Pinto, CE., Rodriguez, D.,
Gomez-Caro, R., De Jong, J., Martinez-A, C., Medema, JP., Hahne, M., APRIL
modulates B and T cell immunity. J. Clin. Invest. 2002.109:1587-1598
Storb, U., Peters, A., Klotz, E., Rogerson, B., Hackett, J Jr., The mechanism of somatic
hypermutation studied with transgenic and transfected target genes. Sem. Immunol.
1996.8:131-140
Strasser, A., Harris, AW., Vaux, DL., Webb, E., Bath, ML., Adams, JM., Abnormalities
of the immune system induced by dysregulated bcl-2 expression in transgenic mice.
Curr. Top. Microbiol. Immunol. 1990.166:175-181
Strasser, A., Whittingham, S., Vaux, DL., Bath, ML., Adams, JM., Cory, S., Harris,
AW., Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and
elicits autoimmune disease. Proc. Natl. Acad. Sci. USA. 1991.88:8661-8665
Stuber, E., Strober, W., The T cell-B cell interactions via OX40-OX40L is necessary for
the T cell-dependent humoral immune response. J. Exp. Med. 1996.183:979-989
Sverremark, E., Fernández, C., Unresponsiveness following immunization with the Tcell-independent antigen dextran B512. Can it be abrogated? Immunology.
1998a.95:402-408
- 56 -
Sverremark, E., Fernández, C., Germinal center formation following immunization with
the polysaccharide dextran B512 is substantially increased by cholera toxin. Int.
Immunol. 1998b.10:851-859
Szakal, AK., Aydar, Y., Balogh, P., Tew, JP., Molecular interactions of FDCs with B
cells in aging. Semin. Immunol. 2002.14:267-274
Szakal, AK., Kosco, MH., Tew, JG., Microanatomy of lymphoid tissue during humoral
immune responses: structure function relationships. Annu. Rev. Immunol. 1989.7:91-109
Szakal, AK., Taylor, JK., Smith, JP., Kosco, MH., Burton, GF., Tew, JG., Kinetics of
germinal center development in lymph nodes of young and aging immune mice. Anat.
Rec. 1990.222:475-485
Takahashi, Y., Cerasoli, DM., Dal Porto, JM., Shimoda, M., Freund, R., Fang, W.,
Telander, DG., Malvey, EN., Mueller, DL., Behrens, TW., Kelsoe, G., Relaxed negative
selection in germinal centers and impaired affinity maturation in bcl-xL transgenic mice.
J. Exp. Med. 1999.190:399-410
Takahashi, Y., Otha, H., Takemori, T., Fas is requires for clonal selection in germinal
centers and the subsequent establishment of the memory B cell repertoire. Immunity.
2001.14:181-192
Takai, T., Ono, M., Hikida, M., Ohmori, H., Ravetch, JV., Augmented humoral and
anaphylactic responses in FcγRII-deficient mice. Nature. 1996.379:346-349
Takeda, K., Kaisho, T., Akira, S., Toll-like receptors. Annu. Rev. Immunol.
2003.21:335-376
Tew, JG., Mandel, TE., Prolonged antigen half-life in the lymphoid follicles of
specifically immunized mice. Immunology. 1979.37:69-76
Tiegs, SL., Russell, DM., Nemazee, D., Receptor editing in self-reactive bone marrow
B cells. J. Exp. Med. 1993.177:1009-1020
Toyama, H., Okada, S., Hatano, M., Takahashi, Y., Takeda, N., Ichii, H., Takemori, T.,
Kuroda, Y., Tokuhisa, T., Memory B cells without somatic hypermutation are generated
from Bcl6-deficient B cells. Immunity. 2002.17:329-339
Tumas-Brundage, K., Manser, T., The transcriptional promotor regulates hypermutation
of the antibody heavy chain locus. J. Exp. Med. 1997.185:239-250
Turner, CA Jr., Mack, DH., Davis, MM., Blimp-1, a novel zinc finger-containing
protein that can drive the maturation of B lymphocytes into immunoglobulin secreting
cells. Cell. 1994.77:297-306
Tuveson, DA., Carter, RH., Soltoff, SP., Fearon, DT., CD19 of B cells as a surrogate
kinase insert region to bind phosphatidylinositol 3-kinase. Science. 1993.260:986-989
- 57 -
Uckun, FM., Burkhardt, AL., Jarivs, L., Jun, X., Stealey, B., Dibirdik, I., Myers, DE.,
Tuel-Ahlgren, L., Bolen, JB., Signal transduction through the CD19 receptor during
discrete developmental stages of human B-cell ontogeny. J. Biol. Chem.
1993.268:21172-21184
Usui, T., Wakatsuki, Y., Matsunaga, Y., Kaneko, S., Koseki, H., Kita, T., Kosek, H.,
Overexpression of B cell-specific activator protein (BSAP/Pax5) in a late B cell is
sufficient to suppress differentiation to an Ig high producer cell with plasma cell
phenotype. J. Immunol. 1997.158:3197-3204
van Egmond, M., Damen, CA., van Spriel, AB., Vidarsson, G., van Garderen, E., van de
Winkel, JG., IgA and the IgA Fc Receptor. Trends. Immunol. 2001.22:545-546
van Eijk, M., Defrance, T., Hennino, A., de Groot, C., Death-receptor contribution to
the germinal-center reaction. Trends. Immunol. 2001a.12:677-682
van Eijk, M., Medema, JP., de Groot, C., Cutting edge: cellular Fas-associated death
domain-like IL-1-converting enzyme-inhibitory protein protects germinal center B cells
from apoptosis during germinal center reactions. J. Immunol. 2001b.166:6473-6476
van Kooten, C., Banchereau, J., Functions of CD40 on B cells, dendritic cells and other
cells. Curr. Opin. Immunol. 1997.9:330-337
van Nierop, K., de Groot, C., Human follicular dendritic cells: function, origin and
development. Semin. Immunol. 2002.14:251-257
van Noesel, CJM., Lankester, AC., van Schijndel, GMW., van Lier, RAW., The
CR2/CD19 complex on human B cells contains the src-family kinase Lyn. Int. Immunol.
1993.5:699-705
van Rooijen, N., Direct intrafollicular differentiation of memory B cells into plasma
cells. Immunol. Today. 1990.11:154-157
Venkataraman, I., Francis, DA., Wang, Z., Liu, J., Rothstein, TL., Sen, R., CyclosporinA sensitive induction of NF-AT in murine B cells. Immunity. 1994.1:189-196
Vos, Q., Lees, A., Wu, Z-Q., Snapper, CM., Mond, JJ., B-cell activation by T-cellindependent type 2 antigens as an integral part of the humoral immune response to
pathogenic microorganisms. Immunol. Rev. 2000.176:154-170
Wagner, SD., Neuberger, MS., Somatic hypermutations of the immunoglobulin genes.
Ann. Rev. Immunol. 1996.14:441-457
→
Wang, D., Wells, SM., Stall, AM., Kabat, EA., Reaction of germinal centers in the Tcell-independent response to bacterial polysaccharide (1 6)dextran. Proc. Natl. Acad.
Sci. USA. 1994.91:2502-2506
- 58 -
Wang, J., Lobito, AA., Shen, F., Hornung, F., Winoto, A., Lenardo, MJ., Inhibition of
fas-mediated apoptosis by the B cell antigen receptor through c-FLIP. Eur. J. Immunol.
2000.30:155-163
Wang, LD., Clark, MR., B-cell antigen-receptor signalling in lymphocyte development.
Immunology. 2003.110:411-420
Wang, Z., Karras, JG., Howard, RG., Rothstein, TL., Induction of bcl-x by CD40
engagement rescues sIg-induced apoptosis in murine B cells. J. Immunol.
1995.155:3722-3725
Weber, JS., Berry, J., Litwin, S., Claflin, JL., Somatic hypermutation of the JC intron is
markedly reduced in unrearranged kappa and H alleles and is unevenly distributed in
rearranged alleles. J. Immunol. 1991.146:3218-3226
Weinshank, RL., Luster, AD., Ravetch, JV., Function and regulation of a murine
macrophage-specific IgG Fc receptor, FcγR-α. J. Exp. Med. 1988.167:1909-1925
Weksler, ME., Innes, JD., Goldstein, G., Immunological studies of aging. IV. The
contribution of thymic involution to the immune deficiencies of aging mice and reversal
within thymopoietin32-36. J. Exp. Med. 1978.148:996-1006
Weng, WK., Jarvis, L., LeBien, TW., Signaling through CD19 activates Vav/mitogenactivated protein kinase pathway and induces formation of a
CD19/Vav/phosphatidylinositol 3-kinase complex in human B cell precursors. J. Biol.
Chem. 1994.269:32514-32521
Wood, C., Fernández, C., Möller, G., Potentiation of the PFC response to thymusindependent antigens by heterologous erythrocytes. Scand. J. Immunol. 1982.16:293301
Wortis, HH., Teutsch, M., Higer, M., Zheng, J., Parker, DC., B-cell activation by
crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways
and results in different B-cell phenotypes. Proc. Natl. Acad. Sci. USA. 1995.92:33483352
Xie, H., Rothstein, TL., Protein kinase C mediates activation of nuclear cAMP response
element-binding protein (CREB) in B lymphocytes stimulated through surface Ig. J.
Immunol. 1995.154:1717-1723
Xie, H., Wang, Z., Rothstein, T., Signaling pathways for antigen receptor-mediated
induction of transcription factor CREB in B lymphocytes. Cell. Immunol.
1996.169:264-270
Yan, M., Marsters, SA., Grewal, IS., Wang, H., Ashkenazi, A., Dixit, VM.,
Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity.
Nat. Immunol. 2000.1:37-41
- 59 -
Yang, X., Stedra, J., Cerny, J., Relative contribution of T and B cells to hypermutation
and selection of the antibody repertoire in germinal centers of aged mice. J. Exp. Med.
1996.183:959-970
Ye, BH., Cattoretti, G., Shen, Q., Zhang, J., Hawe, N., de Waard, R., Leung, C., NouriShirazi, M., Orazi, A., Chaganti, RS., Rothman, P., Stall, AM., Pandolfi, PP., DallaFavera, R., The BCL-6 proto-oncogene controls germinal-centre formation and Th2type inflammation. Nat. Genet. 1997.16:161-170
Yelamos, J., Klix, N., Goyenechea, B., Lozano, F., Gonzalez Fernandez, A., Pannell, R.,
Neuberger, MS., Milstein, C., Targeting of non-Ig sequences in place of the V segment
by somatic hypermutation. Nature. 1995.376:225-229
Yount, WJ., Dorner, MM., Kunkel, HG., Kabat, EA., Studies on human antibodies. VI.
Selected variations in subgroup composition and genetic markers. J. Exp. Med.
1968.127:633-646
Zaitoun, AM., Cell population kinetics of the germinal centers of lymph nodes of
BALB/c mice. J. Anat. 1980.130:131-137
Zhang, J., Liu, YJ., MacLennan, ICM., Gray, D., Lane, PJL., B cell memory to thymusindependent antigens type 1 and type 2: the role of lipopolysaccharide in B memory
induction. Eur. J. Immunol. 1988.18:1417-1424
Zhang, X., Li, L., Jung, J., Xiang, S., Hollmann, C., Choi, YS., The distinct roles of T
cell-derived cytokines and a novel follicular dendritic cell-signaling molecule 8d6 in
germinal center-B cell differentiation. J. Immunol. 2001.167:49-56
Zheng, B., Han, S., Takahashi, Y., Kelsoe, G., Immunosenescence and germinal center
reaction. Immunol. Rev. 1997.160:63-77
Ziegner, M., Steinhauser, G., Berek, C., Development of antibody diversity in single
germinal centers: selective expansion of high-affinity variants. Eur. J. Immunol.
1994.24:2393-2400
- 60 -