It has been almost 100 years since Emil von Behring and Shibasaburo Kitasato
received the first Nobel Prize for the discovery of passive immunotherapy.
In 1888 Emile Roux and Alexandre Yersin isolated a soluble toxin from cultures of diphtheria.
The bacterium itself is only found in the throat, but its destructive effects are found
throughout the body. Clearly, the bacteria must be sending out an invisible factor, most
likely chemical in nature, to cause the body-wide destruction.
This idea was the hypothesis
of Roux and Yersin.
They filtered diphtheria cultures to remove the bacteria and
then injected the remaining fluid filtrate (which we call the supernatant) into healthy
animals.
As expected, the animals showed diphtheria lesions but without any obvious
presence of bacteria.
They then took serum from animals infected with diphtheria and injected it into
healthy animals. When these animals were later inoculated with diphtheria, they were
found to be resistant to infection. We now know this method of conferring infection
resistance as passive immunity.
This first demonstration of defense against infection
was described as mediated by antitoxin. It was clear to von Behring and Kitasato that the antitoxin was specific only for diphtheria; it did not confer any defense
against other forms of infection.
We now know that this antitoxin is composed of antibodies
produced specifically against the diphtheria microbe.
In 1897, Rudolf Kraus first visualized the reaction of antitoxins to bacteria by simply adding serum from
infected animals to a culture of the bacteria and seeing a cloudy precipitate develop as
the antibodies bound the bacteria together.
Other scientists took different approaches and revealed serum-based responses
toward bacteria and their products.
Initially these serum properties were given a range
of different names, such as precipitins, bacteriolysins, and agglutinins. Immunologic
research would have to wait until 1930 before these subtly different properties were
unified and recognized as a single entity.
Long before antibodies were actually isolated
and identified in serum, Paul Erlich had put forward his hypothesis for the formation
of antibodies.
The words antigen and antibody (intentionally loose umbrella terms)
were first used in 1900.
It was clear to Erlich and others that a specific antigen elicited
production of a specific antibody that apparently did not react to other antigens.
Erlich introduced a number of ideas that were later to be proved correct.
He hypothesized
that antibodies were distinct molecular structures with specialized receptor
areas.
He believed that specialized cells encountered antigens and bound to them via
receptors on the cell surface.
This binding of antigen then triggered a response and production
of antibodies to be released from the cell to attack the antigen.
He understood
that antigen and antibody would fit together like a “lock and key.” A different key
would not fit the same lock and vice versa.
However, he did get two important points
wrong. First, he suggested that the cells that produced antibody could make any type
of antibody.
He saw the cell as capable of reading the structure of the antigen bound
to its surface and then making an antibody receptor to it in whatever shape was required
to bind the antigen.
He also suggested that the antigen-antibody interaction took place
by chemical bonding rather than physically, like pieces of a jigsaw puzzle.
Thus, by 1900, the medical world was aware that the body had a comprehensive
defense system against infection based on the production of antibodies.
They did not
know what these antibodies looked like, and they knew little about their molecular
interaction with antigens; however, another major step on the road had been made.
We
can see that the antibody system of defense was ultimately a development of the
ancient Greek system of medicine that believed in imbalances in the body humors.
The
antibody response later became known as the humoral arm of the immune system.
The term humoral (from the Latin word humors) refers to the fluids that pass through
the body like the blood plasma and lymph.
The blood plasma is the noncellular portion
of the blood, and the lymph is the clear fluid that drains via lymph ducts to the
lymph glands and finally into the venous circulation.
These fluids carry the antibodies,
which mediate the humoral immune response.
BASIC STRUCTURE OF ANTIBODIES
Antibodies (immunoglobulins, abbreviated Ig) are proteins of molecular weight
150,000–900,000 kD.
They are made up of a series of domains of related amino acid
sequence, which possess a common secondary and tertiary structure.
This conserved
structure is frequently found in proteins involved in cell-cell interactions and is especially
important in immunology.
Some examples of other members of the immunoglobulin
supergene family are the T-cell receptor; the adhesion molecules intercellular cell
adhesion molecule (ICAM)-1, -2, and -3 and vascular cell adhesion molecule (VCAM);
the coreceptors CD4 and CD8; the costimulatory pairs CD28, CTLA4, B7.1, and B7.2;
and all or parts of many other proteins.
The proteins utilizing this structure are members
of the immunoglobulin supergene family. All antibodies have a similar overall
structure, with two light and two heavy chains. These are linked by both covalent
(disulphide bridges) and noncovalent forces.
One end of the Ig binds to antigens (the Fab portion, so called because it is the fragment
of the molecule that is antigen binding); the other end which is crystallizable, and
therefore called Fc, is responsible for effector functions.
There are five classes (isotypes) of Ig: IgM, IgG, IgA, IgD, and IgE, plus four subtypes
of IgG (IgG1–4) and two subtypes of IgA (IgA1 and IgA2).
Light chains exist
in two classes, Light chains exist
in two classes and K.
Each antibody molecule has either Light chains exist
in two classes or K light chains, not both.
Igs are found in serum and in secretions from mucosal surfaces.
They are produced and secreted by plasma cells, which are found mainly within lymph nodes and which do
not circulate. Plasma cells are derived from B-lymphocytes.
The immunoglobulin molecule consists of two light chains,
each of approximate molecular weight 25,000, and two heavy chains, each of approximate
molecular weight 50,000.
IgA exists in monomeric and dimeric forms and IgM
in a pentameric form of 900,000 kD.
The links between monomers are made by a
J chain.
Additionally, IgA molecules receive a secretory component from the epithelial
cells into which they pass.
This is used to transport them through the cell and
remains attached to the IgA molecule within secretions at the mucosal surface.
The
heavy and light chains consist of amino acid sequences.
In the regions concerned with antigen binding, these regions are extremely variable, whereas in other regions of
the molecule, they are relatively constant.
Thus each heavy and each light chain possesses
a variable and a constant region.
The isotype of an Ig is determined by the
constant region.
L chains are separated from H chains by disulphide (S-S) links.
Intrachain
S-S links divide H and L chains into domains, which are separately folded.
Thus,
an IgG molecule contains three H chain domains, CH1, CH2, and CH3. Between
CH1 and CH2, there are many cysteine and proline residues.
This is known as the hinge
region and confers flexibility to the Fab arms of the Ig molecule.
ANTIBODY RECEPTORS
There are various Fc receptors (R) with different properties.
Monocytes and neutrophils
express receptors (FcR) for the Fc region of IgG.
They constitutively express
FcλRIIa and FcλRIIIa (monocytes) and FcλRIIIb (neutrophils).
Mature tissue
macrophages additionally express FcλRI, as do activated neutrophils.
The principal difference
between these receptors is that FcλRI is a high-affinity receptor (Kd ~ 10-9) and therefore can bind monovalent antibody/complexes, whereas FcλRII and -III are
low-affinity receptors (Kd ~ 10-6) and thus only bind multivalent antibody-antigen
complexes.
SOURCES OF ANTIBODIES
Antibodies are synthesized by lymphocytes. Lymphocytes may be T (= thymus)- processed
or B (= bone marrow)-processed.
Antibodies are made by B-lymphocytes
and exist in two forms, either membrane bound or secreted. B-lymphocytes use
membrane-bound antibody to interact with antigens.
A B-cell makes antibodies all of
the same specificity, i.e., able to react with the same antigenic determinants; its progeny
(as a consequence of mitotic division) are referred to as a clone. The clone will
continue making antibody of the same specificity.
Simultaneously, there will be many
other clones of different specificity. This is known as a polyclonal response.
Antigens
have determinants called epitopes. Epitopes are molecular shapes recognized by antibodies,
which recognize one epitope rather than whole antigen.
Antigens may be proteins,
lipids, or carbohydrates, and an antigen may consist of many different epitopes
and/or may have many repeated epitopes.
B-lymphocytes evolve into plasma cells under the influence of T-cell released
cytokines.
Plasma cells secrete antibodies in greater amounts but do not divide.
They
exist in lymphoid tissues, not blood. Other B-cells circulate as memory cells.
The Life of the B-Cell
B-lymphocytes are formed within the bone marrow and undergo their development
there.
They have the following functions:
1. to interact with antigenic epitopes, using their immunoglobulin receptors
2. to subsequently develop into plasma cells, secreting large amounts of specific antibody, or
3. to circulate as memory cells
4. to present antigenic peptides to T-cells, consequent upon interiorization and processing of
the original antigen.
FUNCTIONS OF ANTIBODIES
Antibodies exist free in body fluids, e.g., serum, and membrane-bound to B-lymphocytes.
Their function when membrane-bound is to capture antigen for which they
have specificity, after which the B-lymphocytes will take the antigen into its cytoplasm
for further processing.
Free antibodies have the functions given below.
Agglutination. Antibodies can agglutinate particulate matter, including bacteria and
viruses. IgM is particularly suitable for this, as it is able to change its shape from a star
form to a form resembling a crab.
Opsonization. Opsonization involves the coating of bacteria for which the antibody’s
Fab region has specificity (especially IgG).
This facilitates subsequent phagocytosis by
cells possessing an Fc receptor, e.g., neutrophil polymorphonuclear leukocytes (polymorphs).
Thus it can be seen that in opsonization and phagocytosis both the Fab and
the Fc portions of the immunoglobulin molecule are involved.
Neutralization. Toxins released by bacteria, e.g., tetanus toxin, are neutralized when
specific IgG antibody binds, thus preventing the toxin binding to motor end plates and
causing persistent stimulation, manifest as sustained muscular contraction, which is the
hallmark of tetanic spasms.
This applies particularly to IgG. In the case of viruses, antibodies
can hinder their ability to attach to receptors on host cells.
Here, only Fab is
involved.
Immobilization of bacteria. Antibodies against bacterial ciliae or flagellae will hinder
their movement and ability to escape the attention of phagocytic cells.
Again, only
Fab is involved.
Complement Activation. Complement activation (by the classical pathway), especially
the Fc region of IgM and IgG, eventually leads to death of bacteria by the terminal complement components, which punch holes in the cell wall, leading to an
osmotic death.
Complement components also facilitate phagocytosis by cells possessing
a receptor for C3b, e.g., polymorphs.
Mucosal protection. This is provided mainly by IgA and, to a lesser degree, IgG.
IgA acts chiefly by inhibiting pathogens from gaining attachment to mucosal surfaces.
This is a Fab function.
Expulsion as a consequence of Mast cell degranulation. As a consequence of antigen,
e.g., parasitic worms, binding to specific IgE attached to mast cells by their receptor
for IgE Fc, there is release of mediators from the mast cell.
This leads to contraction
of smooth muscle, which can result in diarrhea, and expulsion of parasites. Here we
see involvement of both Fab versus parasite antigen, with Fc anchoring the reacting
participants.
Precipitation of soluble antigens by immune complex formation. These consist of
antigen linked to antibody.
Depending on the ratio of antigen to antibody, they can be
of varying size.
When fixed at one site, they can be removed by phagocytic cells.
They
may also circulate prior to localization and removal and can fix complement.
Here Fab
and Fc are involved.
Antibody-dependent cell-mediated cytotoxicity (ADCC).
Antibodies bind to organisms
via their Fab region. Large granular lymphocytes (natural killer [NK] cells) attach via Fc receptors and kill these organisms not by phagocytosis but by release of
toxic substances called perforins.
Conferring immunity to the fetus by the transplantal passage of IgG. IgG is the only
class (isotope) of immunoglobulin that can cross the placenta and enter the fetal circulation,
where it confers immune protection.
This is of great importance to the fetus
in the first 3 months. The precise function of IgD is not known.
It may serve as a maturation
marker of B-lymphocytes.
Primary and Secondary Responses
When we are exposed to an antigen for the first time, there is a lag of several days
before specific antibody becomes detectable. This antibody is IgM.
After a short time,
the antibody level declines.
These are the main characteristics of the primary response.
If at a later date we are reexposed to the same antigen, there is a far more rapid appearance
of antibody, and in greater amounts.
It is of the IgG class and remains detectable
for months or years.
These are the features of the secondary response. If at the same
time we are reexposed to an antigen, we are exposed to a different antigen for the first
time, the properties of the specific response to this antigen are those of the primary
response.
Thus the secondary response requires the phenomenon known as class switching.
This requires cooperation with T-cells of various types, which release cocktails of substances
called cytokines.
These cytokines induce gene rearrangements culminating in
class switching (described below).
This phenomenon is possible because the immune system possesses specific memory
for antigens.
It occurs because during the primary response, some B-lymphocytes,
in addition to those differentiating into antibody-secreting plasma cells, become memory
cells, which are long lived.
GENERATION OF ANTIBODY DIVERSITY
A major question is how antibodies recognize so many different epitopes.
The
antigen-combining site of the antibody molecule is in the variable region of Fab.
Actually,
this site is even more variable than the immediately adjacent sites and is known
as the hypervariable region.
The bond with antigen is of a physical, non-covalent
nature.
As mentioned before, variable (V), and constant (C) regions are genetically encoded.
If we bear in mind that we need to be capable of responding to something on the order
of 1018 antigens, we can appreciate the need for the enormous number of genes necessary
to provide this.
In fact, the amount of DNA that this would involve would be
quite profligate, and nature has solved this problem very ingeniously by a neat little
trick.
In the germline DNA, the V genes encoding the antigen-combining sites need to
combine with the C genes. Additional interposed genes bring about diversity of specificity.
In light chains, these are the J genes, which link V to C, i.e., we have V-J-C.
Joining is imprecise, causing further variation, or combinatorial diversity.
In the case
of H chains, there is yet another region interposed between V and J, the D (for diversity)
gene segment.
Thus, in H chains, we have V-D-J-C, again with combinatorial
diversity.
So, if there are 25
λ light chain V genes, and 5 J genes, constituting light
chain variable regions, there are already 125 possible combinations, disregarding
imprecision of joining.
For K light chains, there are 5 V genes and 70 J genes, yielding
350 combinations.
For H chains, there are 100 V genes, 50 D genes, and 6 J genes,
giving 30,000 combinations.
Overall, disregarding combinatorial diversity, this yields
more than 109 combinations. When we multiply this by joining imprecision, plus a
heightened mutation rate of genes in the hypervariable region, we can see that from
261 genes, we can easily exceed 1018 variations.
The C regions are also genetically encoded, there being four genes for λ light chains,
one for K light chains, and nine H chain C genes (IgM, IgD, IgG1–4, IgA1, IgA2,
and IgE).
IgG is the only class of immunoglobulin capable of crossing the placenta (an Fcmediated
event).
The mechanisms for generating antibody diversity may be summarized as follows:
5. Pairing of heavy and light chains.
Millions of antibody genes come from diverse combinations of gene parts.
Antibodies have a variable region (binding site) and a constant region (holds binding
sites together, interacts with cells).
B-cell maturation joins V (variable), D (diversity),
and J (segments) to form a variable gene region, connected to a constant region. Posttranscriptional
processing removes introns (and extra J regions) to form mRNA.
Class switching changes the constant region type.
Each stem cell produces
an antibody with a different specificity, because it combines a different combination of
V, D, and J exons for both light and heavy chains.
ANTIBODY ENGINEERING YESTERDAY AND TODAY
The discovery of monoclonal antibody (MAb) technology in the late 1970s and
early 1980s opened a new era in human therapeutics. The economic promise of
MAbs was said to be limitless.
In fact, MAbs, could be selected with exquisite specificity.
They were found to orchestrate various components of the immune system such
as ADCC and complement, and they showed a high biologic half-life in blood and tissues,
rendering them effective for prophylactic use.
The toxicity of infused MAbs was
expected to be low because of their biologic nature.
This concept was further supported
by the successful clinical results of mouse antiidiotypic MAbs in the treatment
of lymphoma and leukemias and by U.S. Food and Drug Administration (FDA)
approval in 1986 of the OKT3 and anti-CD3 mouse MAb for acute renal transplant
rejection.
This excess of optimism was soon followed by a period of skepticism after adverse
clinical and laboratory findings with rodent MAbs when they were used clinically in
humans: up to 50% of treated patients developed antimurine antibody responses.
In
addition, the effector functions and biologic half-life were much less efficient.
Adding
to the skepticism were the additional failures of the clinical trials of the anti-lipopolysaccharide
(LPS) mouse IgME5 MAb from Zoma, which was completed between 1992
and 1993, and the human IgM HA-1A (for septic shock) from Stanford/Centocor.
However,
in 1994, the FDA approved the antiplatelet mouse MAb ReoPro to treat the complications
of angioplasty.
This modest success was followed by FDA approval of six
other engineered antibodies between 1997 and 1999.
The resurgence of interest in antibody-based therapeutics was the direct consequence
of the introduction of genetically engineered immunoglobulins and the refinement
of targets for antibody therapy.
MAbs or their recombinant derivatives now
account for the single largest group of biotechnology-derived molecules in clinical trials
and have a prospective market of several billion dollars.
Their applications include
the prophylaxis, therapy, or control of allergic and autoimmune diseases; complications
of angioplasty; sepsis; a variety of inflammatory diseases; many viral and bacterial
infections; organ transplantation rejections; and solid and hematologic tumors.
ANTIGEN-PRESENTING CELLS AND T-CELLS
MHC and Antigen Presentation
Class II major histocompatibility complex (MHC) is an antibody-like protein representing
an extension of the principles by which antibodies are made: MHCs in different
clones have different specificities (like antibodies) but otherwise different structures
and functions.
MHCs have
α and
β chains with binding sites (rather than small and
large chains) and a constant region that anchors the molecule to the plasma membrane
(with a binding site outside the cell).
Cytokines
This is a generic term for messenger molecules (polypeptides) secreted by lymphoid
and nonlymphoid cells that form a mediator network regulating the growth, differentiation,
and function of cells involved in immunity, hematopoiesis, and inflammation.
Cytokines secreted by lymphocytes are also called lymphokines, and those secreted by
monocytes/macrophages are known as monokines.
An interleukin (IL) is a cytokine
that carries a message between leukocytes.
CD 4 helper T-cells are now divided into two subsets based on cytokine profile
and predominant function:
1. Type 1 (Th1) cells produce interferon (IFN)-λ, tumor necrosis factor (TNF)α- and -λ, and
IL-2 (but not IL-4, IL-5, or IL-10) and regulate classical delayed (type IV) hypersensitivity
reactions centered around macrophage activation and T-cell-mediated immunity.
2. Type 2 (Th2) cells elaborate IL-4, IL-5, IL-6, and IL-10 and participate in immediate (type
1) hypersensitivity reactions and B-cell antibody-mediated immunity.
Proinflammatory cytokines, such as TNF (α and
β) and IL-1, which are produced
by activated macrophages, mediate local and systemic effects, including the induction
of the acute-phase reactions of inflammation.
Chemokines (chemotactic cytokines) belong to a family of low-molecular-weight
proteins (with complex names/eponyms) that are secreted by monocytes (e.g., monocyte
chemotactic protein [MCP]), macrophages (e.g., macrophage inflammatory protein
[MIP]), and T-cells (e.g., regulated upon activation normal T-cell expressed and
secreted [RANTES]) that influence leukocyte motion and that attract leukocytes to sites of tissue inflammation or infection.
Surprisingly, in HIV infection, specific
chemokine receptors on CD4 target cells are now known to function as coreceptors
required for viral entry.
B-CELLS
Early Development of the Repertoire
The evolutionary selection pressure guiding T-cell and B-cell repertoire development
is the same in each case: to generate a range of specificities that will protect against
various and unpredictable infectious disease challenges while limiting the potential for
reactivity against self.
This selection pressure acts on the level of the individual animal,
such that the individual with the most effective repertoire in a particular time and
place is most likely to survive and reproduce.
The selection pressure also acts on the
level of the population, such that repertoire diversity maintained within a population
makes it more likely that some individuals will survive to reproduce after an infectious
outbreak.
The downside of clonal deletion as a mechanism for tolerance is that it creates holes
in the repertoire. A pathogen could take advantage of these holes by mimicking self to
evade immune recognition.
For T-cells, this problem is dealt with by balanced polymorphism
of MHC within a species. T-cell recognition of peptide in the context of
polymorphic MHC molecules provides each individual with a different T-cell repertoire
complete with different holes.
Thus MHC polymorphism provides protection against disease at the level of the population. Because B-cells recognize native antigen,
and most of us express the same set of native proteins, any holes in the B-cell
repertoire created by clonal deletion would be the same across the population,
putting the entire population at great risk from infectious agents that mimic “self”
proteins.
Whereas the recognition of polymorphic MHC by T-cells protects populations
from this sort of threat, B-cell recognition of native antigen precludes a similar
strategy.
Antigen Recognition and Lymphocyte Development
B-cell development differs significantly from T-cell development in that negative
selection of autoreactive B-cells can occur in the same microenvironment in which productive
immune responses begin, the outer T-cell zone of the spleen.
The maturation
of B-cells in this more public environment has important implications for the mechanisms
that maintain self-tolerance and contribute to the development of autoimmunity.
This type of development allows for the shaping of the B-cell repertoire with multiple
specificities, including weakly autoreactive and crossreactive specificities, into the functional
repertoire.
The evolution of the humoral immune system was challenged by having
on hand as diverse an array of antibody-producing cells as possible to address the
multiple types of invaders discussed earlier.
Much of T-cell development occurs in the thymus, geographically sequestered from
the sites of active immune responses.
This cloistered environment ensures that many
self-reactive T-cells are eliminated before joining the mature T-cell repertoire. B-cells
also undergo several forms of negative selection of self-reactive specificities.
Recent
experiments suggest that, in contrast to T-cell development, much B-cell negative selection
occurs in the same location in which immune responses to foreign antigens are
initiated the outer T-cell zone of the spleen.
This maturation of B-cells in a public environment has important implications for the mechanisms that
maintain self-tolerance and that might contribute to the development of autoimmune
disease.
Here, we suggest that the public shaping of the B-cell repertoire allows the
recruitment of multiple specificities, including weakly self-reactive specificities, into
the functional immune repertoire and that this mechanism for increasing repertoire
diversity offsets the risk of autoimmunity.
B-cell selection, like T-cell selection, functions to balance the need for repertoire
diversity with the need to protect against autoimmunity.
T-cells and B-cells recognize
antigen in fundamentally different ways, and these differences in recognition are
reflected in differences in the mechanisms of repertoire generation.
T-cell recognition
is inexorably associated with recognition of self.
T-cells recognize peptide antigen
complexed with MHC molecules, constraining recognition to antigen processed and
presented by cells.
T-cell selection reflects this recognition by allowing only
those T-cells with receptors that bind MHC to mature, while eliminating those T-cells
that strongly bind self-peptide MHC complexes during development in the thymus.
Signals to the T-cell that stimulate activation of T-cell immune responses in
the periphery induce deletion of maturing, self-reactive cells in the thymus.
Thymic T-cells that have yet to complete development and selection are prevented from
joining the functional immune repertoire; the cloistered environment of the thymus
thus protects against autoimmunity.
In contrast to T-cell recognition, B-cells recognize native antigen that is not necessarily
associated with cells.
B-cell development also begins in an isolated environment
in the bone marrow, where high avidity self-reactive B-cells are deleted.
Although it was generally thought that most B-cell-negative selection occurred in the
bone marrow, several lines of evidence point to a key distinction from T-cell development.
First, the bone marrow appears to export a larger proportion of the B-cells that
it produces than the thymus.
These newly exported B-cells are relatively immature
cells that migrate from the bone marrow to the outer T-cell zones of the white pulp
of the spleen.
These newly emigrated splenic B-cells express high levels of the
heat-stable antigen (HSA), a maturation marker common to developing B- and
T-cells.
By contrast, HSAhi T-cells are found only in the thymus, as maturing
T-cells lose HSA expression before migrating to the periphery.
Second, when
the recirculating B-cell repertoire has attained an adult size and steady state, only a
small fraction of these recent bone marrow emigrants persists after reaching the splenic
T-cell zone.
The cells that do persist have a skewed V-region repertoire.
This splenic restriction point in B-cell production eliminates unwanted B-cells
by the same order of magnitude as occurs for T-cells exclusively in the thymus.
A key
question is whether immature B-cells are selected against within the splenic T-cell zone
because they fail a positive selection step for particular specificities or because they
trigger a negative selection step against particular specificities.
The first evidence that immature B-cells are negatively selected in the spleen came
from Cyster et al, who showed that self-reactive B-cells recognizing circulating
lysozyme antigen accumulate in the T-cell zone of the spleen and are excluded from
migration into the B-cell follicles, with an efficiency that is directly proportional to the
level of self-ligand present, the affinity of the receptor, its signaling properties, and the
presence of competing B-cells.
Self-reactive cells that are excluded from
the follicular recirculating repertoire are short lived (1–3 days), whereas, cells that
enter the B-cell follicles are long lived and recirculate for 1–4 weeks.
The significance of this follicular exclusion checkpoint in negative selection of selfreactive
B-cells has recently been extended by studies tracking the development of
B-cells specific for double-stranded DNA (anti-dsDNA), a clinically important specificity,
in the context of a polyclonal B-cell repertoire.
Mandik-Nayak et al. have shown that prototypic anti-dsDNA B cells are not deleted in the bone marrow but are
exported to the spleen as relatively immature cells with a short half-life relative to
the bulk of the repertoire.
They also show that these autoreactive cells localize to the
interface between the B-cell and T-cell zones of the spleen.
Together with the lysozyme
model antigen data, and the evidence that many immature cells are competitively
selected against at this site, it seems likely that B-cells bearing many different autoreactive
specificities will join the peripheral B-cell population and be subject to selection
at this stage and site within the spleen.
The exclusion of newly produced autoreactive B-cells from the B-cell follicles
places these potentially pathogenic cells in a site known to be important for the initiation
of antibody responses to foreign antigens the outer T-cell zone.
Indeed,
autoantibody-producing cells in autoimmune mice appear and accumulate in the outer
T-cell zone, and it has been proposed that the pathogenic autoantibody production
results from a failure of B-cell tolerance in this site.
Self-reactive cells that are excluded from follicles are also functionally anergic that is, signaling by their B-cell
receptors (BCRs) is reversibly altered so that they make weak mitogenic responses to
antigen.
Nevertheless, antigens with high avidity binding can deliver strong signals
to the B-cells that partially override anergy and induce modest proliferation and
antibody production by maturing self-reactive B-cells.
Thus self-reactive B-cells
that have yet to complete development and negative selection might be recruited into
the functional immune repertoire if they crossreact avidly with a foreign antigen; the
public environment of the spleen seems to encourage this recruitment at the risk of
autoimmunity.
Why risk autoimmunity by requiring so much of B-cell-negative selection
to occur where immune responses begin?
The Autoimmune Solution
The export of self-reactive short-lived cells into a splenic B-cell pool, in which lifespan
is inversely proportional to the degree of self-reactivity, might solve the problem
of holes in the B-cell repertoire, much as MHC polymorphism serves to solve the hole
problem in the generation of the T-cell repertoire.
In any one individual in a population,
at a particular time, a proportion of the B-cell repertoire is contained in the shortlived
B-cell pool, being excluded from entry into the B-cell follicles.
In the absence of
infection, self-reactive cells within this population will die within a few days and so
pose little risk of causing a pathogenic autoimmune response.
Autoimmunity is also
avoided by requiring stronger signals to recruit autoreactive B-cells into an immune
response than are required to recruit naive B-cells and by producing smaller bursts
of progeny when autoreactive cells clear the higher activation hurdle.
Because of the huge potential B-cell repertoire encoded in the genome, the actual B-cell repertoire
available at any one time is likely to differ between individuals based on the probable
recombination and expression of BCRs.
Accordingly, each individual within a population
will express a different B-cell repertoire, with varying propensity toward autoimmunity
when an infectious agent appears.
The repertoire diversity provided by the short-lived pool of B-cells might work in
concert with the probable differences in B-cell pool composition between individuals
to ensure that some individuals will mount effective B-cell responses against an infection.
This solution to plugging the holes in the repertoire might be buttressed by the
unique ability to fine-tune B-cell specificity further, by hypermutation and additional
rounds of negative selection in germinal centers.
The independent processes of anergy
and negative selection in germinal centers might account for why these modest autoantibody
responses do not achieve high concentrations and do not normally exhibit sustained
or recall characteristics.
The effectiveness of this system depends on the availability of a diverse pool of
B-cells within each individual at any one time, as well as differences in pools between
individuals.
Whereas T-cell deletion in the thymus helps to protect against selfreactivity
within the T-cell repertoire, the inherent short lifespan and more rigorous
signaling requirements of self-reactive B-cells helps to protect against self-reactivity
within the B-cell repertoire.
Whereas MHC polymorphism provides diversity in T-cell
repertoires within populations, the probable generation of BCR specificities and the
short-term inclusion of weakly self-reactive specificities might provide diversity among
the B-cell repertoires within populations.
Seen in this light, there might be a clear advantage to transiently maintaining weakly self-reactive B-cells in the periphery,
where they can potentially contribute to an acute immune response to infection.
CONCLUSIONS
The presence of nonpathogenic anti-self antibodies and antibodies derived against
the normal bacterial flora colonizing the vertebrate host in the serum of normal individuals
and their non-anamnestic rise and fall during immunization provides evidence
that self-reactive B-cells that secreting IgM and in some cases IgG autoantibodies exist
and are activated in the peripheral B-cell pool.
One source of these relatively
low-avidity autoantibodies is likely to be activation of short-lived B-cells in the outer
T-cell zone by high-avidity foreign antigens.
The relative contribution of these preexisting
reactive B-cells to total repertoire diversity is not known; however, their influence
on disease resistance and susceptibility are profoundly observed during the
parasitic infection known as leishmania in mice.
Experimental leishmaniasis offers a well-characterized model of Th1-mediated control
of infection by an intracellular organism.
Susceptible BALB/c mice aberrantly
develop Th2 cells in response to infection and are unable to control parasite dissemination.
A previously identified antigen, Leishmania homolog of receptors for activated
C kinase (LACK), was found to be the focus of this initial response.
The early CD4
T-cell response in these mice is oligoclonal and reflects the expansion of memory,
Vβ4/Vα8-bearing T-cells in response to the LACK antigen.
It appears the T-cells were
initially derived to a specific and crossreactive antigen found on a bacterial species colonizing
the mouse gastrointestinal tract during its early lifetime.
IL-4 generated by
these cells is believed to direct the subsequent Th2 response.
Mice made tolerant to
LACK by the transgenic expression of the antigen in the thymus exhibited both a
diminished Th2 response and a healing phenotype.
Thus, T-cells that are activated early
and are reactive to a single antigen play a pivotal role in directing the immune response
to the entire parasite.
Thus, breakthroughs in our knowledge of humoral immunity may be coming with
our understanding of its development during differentiation and initial repertoire development
as the host establishes itself in the environment.
It seems that successful
pathogens may have explored these subtle overlaps between self and the normal colonizing
flora, which in a distant way is part of self in that they permit the survival of
the host through numerous important symbiotic mechanisms.
