Download Immunology 5

Immunology 5 – B lymphocytes
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Why are B and T lymphocytes relevant?
Their absence results in an inability to fight infections.
B and T lymphocytes are said to operate during adaptive immune responses. But what
are adaptive immune responses? Responses tailored towards fighting a particular
pathogen by means of the specificity between the B cell receptor or the T cell receptor
towards the antigens displayed by that pathogen. Adaptive immunity is evolutionarily
more recent than innate immunity. Innate immunity which is activated within minutes of
encountering a given antigen serves to not only buy time while the adaptive system is
galvanized into action but it also forms an important part in actually secreting certain
soluble mediators which can increase lymphocyte production and by displaying the
antigens on MHC Class 2 receptors by particular professional antigen presenting cells.
All of these responses result in the activation of the adaptive immune system which is
responsible and capable of halting most infections and killing the pathogen involved by a
number of ways. The b and the t lymphocytes, in essence, form the effector arms of
adaptive immunity.
Humoural immunity is largely determined by B cells. They are responsible for the
production of antibodies; the topics for discussion in the lecture prior to this, and the cell
mediated immunity is handled by the T lymphocytes.
B lymphocytes, in summary, are white blood cells responsible for the production of
antibodies and some of them also form memory cells following the initial exposure of a
pathogen, which allows for a quicker, faster and more effective response on second
encounter with the particular pathogen.
B cells are derived from haematopoietic stem cells in the bone marrow. They, unlike the t
cells, also develop and mature within the bone marrow.
Following this, they migrate into the circulation, blood and lymph circulation, and can
act and carry out their respective functions in this manner.
B cell generation and maturation actually occurs in the bone marrow in the absence of
any antigen. But antibody production is a strictly antigen-mediated process, B cells must
be activated in the presence of an antigen before they can produce antibodies, which
form the effector arms of adaptive immunity along with the T cells.
The latter part occurs in the lymphoid organs.
In the bone marrow, in the absence of antigen, a pro B cell becomes a pre B cell which
becomes an immature B cell which becomes a mature B cell.
The B cell specificity lies in the B cell receptor, which includes a surface immunoglobulin
molecule, and we find that each BCR is specific for interaction with only a particular
antigen.
Many identical B cell receptors are present on the surface of a given B cell conferring
specificity towards a particular antigen.
BCRs have particular antigen binding region, which encapsulates the immense diversity
seen in these receptors.
The structure of the b cell receptor
The B cell receptor is a transmembrane complex with certain sections of the protein
projecting out towards the extracellular environment and some regions remaining inside,
on the cytosolic surface of the cell.
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It is composed of mLgG along with di-sulphate linked heterodimers known as lg alpha
and beta.
The latter components are necessary for the efficient functioning of the BCR because
they have cytoplasmic tails long enough for the signal transduction necessary
compensating for the short cytoplasmic tail of the main immunoglobulin molecule.
Note that the latter molecules are also part of the immunoglobulin supergene family and
therefore have immunoglobulin-like folds and domains.
Immunoglobulin Genes and the process of re-arrangement
As mentioned before, the immunoglobulins have two chains, the light and the heavy
chains.
Each chain has a variable and constant region.
Instead of being encoded by a single contiguous DNA sequence, the immunoglobulin
polypeptide chains are encoded by a set of gene segments or more accurately, sets of gene
segments. These gene segments can be selected and re-arranged and joined together in
various ways during B cell development and it is this process of recombination which is
responsible for what is known as the generation of diversity in the B cell receptors. As
we will see shortly in the next lecture, the diversity of the T cell receptor is developed in a
similar way. It should also be noted that this process of gene segment re-arrangement and
joining seems to be entirely specific to the immune system.
These gene segments include Joining and Diversity genes in addition to the Variable and
Constant genes. J, D and V will eventually unite to form one functional gene encoding
the variable segment of the chain.
Each given set of segments is variety of genes which encode for that particular piece of
gene.
To understand this better, one can imagine there being for example a number of leader
genes, a number of joining genes, a number of diversity genes and so on. One can
immediately see the simply logic of arranging genes in this manner. We can generate an
enormous, almost unlimited diverse array of different functional genes encoding for the
variable regions which allows the human immune system as a whole to have a vast
repertoire of B and T cells which specificities for almost every antigen possible.
During the development of the B cells, the gene segments are brought closer together, rearranged and then joined in a particular order. The process of re-arrangement is known
as somatic recombination and occurs in the complete absence of antigen to generate
that aforementioned repertoire.
Once the complete light and heavy chain genes have assembled, they can be transcribed
and translated to assemble the B cell receptor, the surface immunoglobulin.
Alternatively, the molecule can be separated from the B cell and secreted into the plasma
as soluble antibody. Note that the process of gene re-arrangement and subsequent joining
in new configurations is exactly the same for immunoglobulins destined for the surface of
the B cell or destined to be released into the fluid surrounding cells, etc.
After the initial gene re-arrangements have taken place, the entire gene is transcribed
including the exons and the introns. The introns are then spliced by means of
spliceosome and the resulting processed mRNA can be translated into protein. The
leader peptide sequence is then removed by proteolytic enzymes. This brings the VJ
segment or VDJ segment (in the case of heavy chains close to particular Constant chain
mRNA)
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The V region of light chains is composed of V and J segments but heavy chains have V,
D and J segments. In order for a complete V region to be transcribed the V and the J
regions, for a given light chain, must be cut out of the germline DNA by means of certain
enzymes, discussed later, and then rejoined in a particular unique configuration, certain
enzymes are once again, involved. For example one J segment from a range of J
segments is selected and combined with one V segment from a range of V segments. For
the heavy chains, a similar selection occurs for the V, D and J segments resulting the in
the completion of a complete Variable Heavy gene.
The complex of enzymes involved in the somatic recombination process is known as
VDJ recombinase. The enzymes are responsible for the cleavage and rejoining of the
DNA involved in re-arrangement.
Two of these enzymes, RAG1 and RAG2 are responsible for the first cleavage step.
These enzymes are only found in lymphocytes.
Human light chain synthesis
There are two types of light chains in antibodies which can be found in ALL the five
classes of antibodies, the kappa and the lambda chains. The gene locus for the kappa
chain is contained on a particular locus of chromosome 2. In the germline of humans,
there are approximately 30 different V kappa genes found in the kappa locus of
chromosome 2. We find that each V kappa genes encodes from 1 to 95 amino acids
stemming from the N region of the polypeptide. Downstream of the V kappa genes, we
can find the J kappa genes. There are, of course, a number of J kappa genes but EACH
one of them encodes for amino acids 96-108 of the kappa variable region. After a long
intron section, the locus ends in one C kappa exon, which is responsible for encoding the
constant region of the Kappa light chain.
In order to synthesize a kappa light chain, a cell early in the B lymphocyte lineage selects
a particular V kappa exon from a selection of V kappa exons and after a process of DNA
re-arrangement catalyzed by VDJ recombinase, joins it to a particular J segment from a
selectrion of J segments.
The intervening DNA segment is looped out and removed, destined for ultimate
degradation.
From this re-arranged DNA, a primary RNA transcript is made.
This primary RNA transcript then undergoes splicing in order to remove the intronal
sections between the coding parts of the mRNA. This results in the particular V,J and C
exons being brought together as one continuous linear section of nucleic acid.
This RNA is now ready for translation in the cytoplasm.
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The process is similar to the lambda chains except that lambda chain loci are found on
chromosome 22 in humans and there are about 30 variable lambda genes as well as four
joining lambda genes, with each particular J segment being associated with a different
constant lambda section. One can, therefore, infer that there are in fact four different
classes of the lambda light chain in humans, depending on the variety conferred by the
four different constant chains.
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Human heavy chain synthesis
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In the human genome, there are approximately 50 variable, 25 diversity, and six joining
segments all involved in the encoding of the variable region of the heavy chains. In other
words, there is an inclusion of another segment as compared to light chains, the diversity
segment, which has been to shown to code for third hypervariable region or the
complementarity determining region.
The mechanism for heavy chain synthesis, otherwise, is quite similar to light chain
synthesis.
First the D and J segments are joined.
Then the V segments joins to the completed DJ segments.
The C region exons are brought closer together to VDJ segments by means of the RNA
splicing introns between the respective exons.
Note the abundance of different kinds of constant chains in the DNA. This is the basis
for the five different antibody classes. The DNA can be re-arranged so that the complete
VDJ segment can associate with different constant region by means of isotope switching
or class switching.
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The different Ch receptors are responsible for different effector functions. This has been
discussed in detail in the last section.
Now that we have discussed the various processes involved in light and heavy chain synthesis,
let us look quickly over the various ways in which diversity is generated as a whole in this
process and related processes.
1. V, D and J segments are present in multiple different copies in the germline DNA. This is
referred to, not surprisingly, as germline diversity.
2. VJ and VDJ segments can recombine in different ways and in multiple combinations.
Due to the presence of multiple copies of the individual segments and the number of
ways in which they can associate, we have a vast number of different combinations
which can result. This is known as combinatorial diversity.
3. The formation of the junction between the V and the DJ segment involved DNA
cleavage followed by the addition and subtraction of nucleotide to create a viable joint.
As a result, different coding sequences can be created at the joint regions in different
antibodies leading, once again, to much greater diversity. This is done through the
random addition of nucleotides; the enzymes is responsible is terminal deoxynucleotidyl
transferase or more simply, TdT.
4. Multiple combinations can occur between the light and heavy chains. This allows for a
number of various combinations and as a result, we have a number of different antibody
specificities.
5. Somatic hypermutation after encounter with the antigen can also function as an
additional technique to generate an even better fit to the antigen and allowing for an
increased affinity of the antigen to the antibody.
The diagram demonstrates a protypical gene which encodes for a given membrane protein. This
is important because of its obvious value in the synthesis of the B cell receptor, which does not
consist only of the immunoglobulin, but also two other components, known as lg alpha and beta
which exist in conjunction with the main immunoglobulin. They have longer cytoplasmic tails
for efficient signalling, compensating for the relatively short cytoplasmic tail of the main surface
immunoglobulin; not useful for signal transduction.
Note also the synthesis of a transmembrane region composed largely of hydrophobic amino
acids, which helps to hold the transmembrane protein in place, as a result of hydrophobic
interactions with the components of the lipid bilayer.
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Adaptive immune responses are characterized by their specificity, diversity and
memory.
The basis of the adaptive immune response is clonal selection.
If we review at this point, we find that the stem cells present in the bone marrow are
subject to the generation of diversity, the VDJ recombinase enzymes are responsible for
the formation and re-arrangement of the B cell DNA. The primary RNA transcript is
synthesized and we find that VD, J and C segments can be effectively unified by splicing
out of the introns in between the coding sections. This results in the formation of one
complete, uninterrupted
polypeptide
chain, this is
synthesized
and then the
process
also
happens for the
light
chains.
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(In actual fact, the heavy chain is synthesized first in the presence of a surrogate light
chain and then the light chain is produced). At the end of this process, we have a B cell
which is specific to a particular antigen, but it is still termed as an immature B
lymphocyte. The last point of maturation is where the self-reactive B lymphocytes are
destroyed.
This is ultimately one of the trade-offs of such an incredibly diversity-generating process;
there is absolutely no way of ensuring that some B cells will be produced which are
reactive against the cells of the body. If these cells are not eliminated, then the immune
response will be misdirected against the cells of the body, and then we’d be in trouble.
Nevertheless, this process of elimination of self-reactive B lymphocytes is not fully
perfect, as is demonstrated by the continued existence of autoimmunity.
Clonal selection occurs when at this point, the mature B lymphocytes in the blood and
the lymph encounter an antigen with a number of epitopes. Chances are that a number of
B lymphocytes will each recognize a particular epitope of the antigen. This binding of the
antigen to the BCR can bring about activation of the B cell which will then proliferate,
produce clones and differentiate into plasma cells, tasked with producing large amounts
of soluble antibody specific to that particular epitope, and memory cells which remain in
the blood for a quicker response on a repeated exposure to that particular antigen.
It should be noted, however, that B cells cannot be activated by exposure to antigen
alone. They MUST be provided co-stimulation by either T helper cells or by certain
microbial constituents or specialized accessory cells.
The use of the T helper cells is what is known as Thymus dependent activation of B
cells. This occurs for all the antibody classes and results in the production of memory.
This occurs when the BCR binds to its complementary antigen. The receptor antigen
complex is internalized, and enclosed within an endocytic vesicle where it fuses with a
lysosome. The antigen is degraded into peptides, assembled along with MHC Class 2
molecules and displayed on the surface of the B cell. The T cells are now able to
recognize this bound peptide antigen if they have the specific T receptor for the job and
they secrete certain cytokines which bind to certain receptors on the B cells as a result of
which the B cell enters the cell cycle, proliferates, produces a clone of identical B cells
with the identical receptors, differentiates into memory cells and plasma cells. Hey
presto, we gat a-bodies!
However, there is also a means of Thymus independent activation by means of costimulation being provided by the microbial constituents or other specialized accessory
cells. This latter form only encompasses lg M and does not result in the development of
immunological memory. In Di george syndrome, individuals lack a thymus and T cells
but they are still capable of producing antibody by this pathway.
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B cell proliferation is under the control of specific cytokines but under the influence of
certain cytokines, class switching is said to occur.
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Final note on Immunological memory. The clonally expanded B and T cells produce as a
result of the first encounter with the antigen diffentiate partly into memory cells, which
also turn over in fact. These memory cells can effectively eliminate the pathogen on
subsequent re-entry and this is what allows for a heightened, quicker and more effective
response to the pathogen. This phenomenon allows for life-long immunity to certain
pathogens, the memory cells can always protect the individual.
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Note the lg G is involved more actively in the secondary response whereas lgM predominates in primary response.
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Note also that antibody affinity increases with the duration of an infection and also with
repeated infections.
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Read polyclonal and monoclonal antibodies from the books or lecture notes. Important
factors to note are the hybridoma method for the production of monoclonal antibodies
and their immense utility in diagnosis, treatment, etc.
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