So B-cells, as their name suggests, ‘B’, develop
in the bone marrow. So the bone marrow is where
B-cells develop. And in fact, virtually all the
cells of the immune system start of from multipotent
hematopoietic stem cells in the bone marrow. And
B-cells can become fully mature within the bone marrow.
They don’t need to go anywhere else to become
mature B-cells, they develop within the bone marrow.
Once they are mature, they can leave the bone marrow
and travel to the secondary lymphoid tissues;
the mucosa-associated lymphoid tissue, the lymph nodes
and the spleen. So let’s have a look at how
B-cell development in the bone marrow occurs and
the stages in B-cell maturation in the bone marrow.
I’ve already mentioned. They start off from
hematopoietic stem cells. These develop
into pro-B cells. Those hematopoietic stem cells,
instead of becoming a mast cell, or instead of
becoming a dendritic cell, those that are going to
become B-cells initially become pro-B cells.
The next stage in their development is to become a
pre-B cell. Then they develop into immature B-cells
and of course eventually into mature B-cells.
There are two stages in this development process
where these B-cells undergo very, very extensive
cell division, in other words proliferation.
That’s at a very early stage in their maturation,
at the stem cell, pro-B cell stage.
And then later on, at the pre-B, immature B-cell
stage, they undergo further massive rounds of
proliferation. And they’re dividing all the time,
but these two stages in their development are
characterized by particularly extensive cell
proliferation. There are two genes that are called
RAG1 and RAG2. Stands for Recombination Activating
Genes. We already mentioned that the immunoglobulin
genes in the B-cell undergo recombination to create
diversity. These Recombination Activating Genes
obviously play a role in that as their name suggests.
And these genes are also expressed at two different
points in the development and maturation of B-cells,
at the pro-B, pre-B stage. And then they can become
re-expressed later on in immature B-cells, to
fine tune the antigen receptor on the B-cell.
There’s another molecule which we’ll hear about
shortly called TdT. Terminal Deoxynucleotidal Transferase.
Bit of a mouthful that isn’t it?
We normally just refer to it as TdT because
Terminal Deoxynucleotidal Transferase is quite a long
word to say. So, TdT. And that’s expressed again
at the stage between pro-B cells and pre-B cells.
If we look at the immunoglobulin gene DNA
in the B-cell and the RNA that’s produced from that
DNA. In the stem cell stage and the pro-B cell stage,
these immunoglobulin genes are what we call, in the
germline configuration. That means how these genes
are inherited through the sperm and the egg, the
germline, and every single cell in the body
will have this DNA, just like you have genes for
all the different genes you have in all the
different cells. And in all the cells in the body,
they will be in the germline orientational configuration.
And it’s only when cells develop to become B-cells
that these immunoglobulin genes recombine.
And this recombination initially starts off at the
pre-B cell stage, and some segments within the
heavy chain genes for the immunoglobulins.
Because immunoglobulins consist of a heavy chain
and a light chain. The first event that says,
“Mmm, that cell, that cell’s going to become a B-cell”,
is when the B-cell recombines its heavy chain genes.
and it recombines three gene segments as we’ll
see shortly. They’re called V, D and J. Variable,
diversity and joining. Once that recombination
process has occurred in the pre-B cell, messenger
RNA is produced for the heavy chain and that includes
a segment that is from the constant region called
mu(µ). So you get µ mRNA in the pre-B cell.
As that cell then develops to the final stage before
full maturity, in other words the immature B-cell,
the recombined heavy chain gene associates the
product of that gene, associates with the product
of a recombined light chain gene. In the light
chains, the V gene segments and J gene segments
that recombine produce a light chain protein. So now
we have Ig for the DNA that’s recombined in the
heavy chain and the light chain. We have a µ heavy
chain protein and either a kappa(k) or lambda(λ)
light chain protein produced from the k or λ
messenger RNA. And then finally we end up
via alternative splicing of the V, D and J with
the constant region of the IgM antibody which is
encoded by a Cµ gene or the IgD antibody encoded
by a Cδ gene. So the stage of maturation.
Early on the immunoglobulin genes are not recombined,
so there’s no expression. In order to express
these genes they need to be recombined. They only
recombine in B-cells. And they only recombine
at the pre-B cell stage. So you start off at the
pre-B cell stage with the first indication that this
cell is going to become a B-cell. It has cytoplasmic
µ heavy chain and on the surface it has what is called
the pre-B cell receptor, which is a surrogate light
chain. It’s not the proper light chain but it’s a
sort of intermediate light chain that’s produced
to put together with the µ heavy chain.
And then once the light chain genes have been
recombined, you get the full mature antibody
molecule on the surface of this immature B-cell.
So it’s a mature antibody but it’s on the surface
of an immature B-cell, and this is membrane IgM.
And then as well as IgM, another class of antibody,
the IgD class is put on the surface of the mature
B-cell. As well as having antibodies on their cell
surface, B-cells have thousands of other molecules
on their cell surface just like every cell does.
And we can use certain molecules on the surface of
B-cells to identify which stage in maturation
they are. So for example, the molecule CD34 is
present on the surface of the stem cells.
It’s also present on the surface of the pro-B cell.
But additional molecules become expressed,
such as CD19 and CD10. And then as those pro-B cells
develop into pre-B cells, there is low expression
of a molecule called B220. But as that pre-B cell
develops into an immature B-cell, the expression of
CD34 is lost. So if a cell has a molecule on its
surface, we describe it as a CD-whatever
positive cell. If it doesn’t have expression, we
describe it as a CD-whatever negative cell.
So in other words, the stem cells, the pro-B
cells and the pre-B cells are CD34 positive,
whereas the immature B-cell is CD34 negative. At
that stage of the immature B-cell, there is a low
level expression of IgM antibodies on the surface of
the B-cell. And then as the B-cell further matures
to become a fully mature B-cell, the level of
expression of IgM increases. So where are these
different types of B-cell, these different stages of
maturation found? Well the stem cells, the pro-B
cells, the pre-B cells are all within the bone marrow.
And then as these cells develop into immature B-cells,
they can leave the bone marrow and enter what
immunologists refer to as the periphery;
rather strange term that, the periphery. Simply
means anywhere outside of the primary lymphoid tissues.
So anywhere in the body that isn’t the bone marrow or
the thymus, immunologists refer to as the periphery.
And the mature B-cells are found in the periphery,
or move to the periphery. The response to antigen
clearly if the B-cell doesn’t have an antigen
receptor on its surface, it can’t respond,
it has no way of responding to antigen. So because
there is no surface antibody on stem cells,
pro-B cells or pre-B cells, there’s no response to
antigen. At the immature B-cell stage, when there is
low level expression of IgM, the B-cells can undergo
what is called negative selection or deletion.
And this refers to the fact that if those B-cells
encounter molecules from our own body, what we call
self-antigen, then they become deleted, because we
don’t want B-cells that can attack our own body
components. We want B-cells that can fight infection
coming from outside the body. So there’ll be deletion
of B-cells that recognize self-antigen and there can
be receptor editing. And this is why RAG1 and RAG2
get re-expressed at this stage. So that the receptor
can be changed. If it reacts with self, it can be
changed to a different receptor that reacts with a
foreign pathogen rather than reacting with self.
And then once they are mature, of course the B-cells
are fully able to respond to antigen. I mean that’s how
it should be obviously. You need the B-cells to be
able to respond. And they can become activated,
they can proliferate, divide up in number and
they can differentiate and become specialized.
Particularly become specialized to produce a
progeny of a B-cell that is called a plasma cell.
And what plasma cells do is they secrete very large
amounts of antibody of the same specificity that acts
at the B-cell receptor on the surface of the B-cell.
So, in the bone marrow we have these pre-B cells,
this early stage of development. And they have on
their cell surface not a mature form of the antibody
molecule but a preliminary version if you like of
the antibody molecule where you have the normal
version of the IgM heavy chain which is called
µ heavy chain. But it’s not a mature version
of the light chain. They have what we refer to as a
surrogate light chain, and this is made up of two
components. One is called V pre-B and the other is
called λ5. And then as the pre-B cell matures
into an immature B-cell, it swaps the surrogate
light chain for a proper, a real light chain.
And that can either be the k light chain or the
λ light chain. So that the mature B-cell now has
on its cell surface a proper mature version of the
antibody molecule. And initially these mature B-cells
that have not yet encountered antigen, we call them
naive B-cells. They’ve not yet met antigen at all.
They express on their cell surface two different
classes of antibody. IgM, surface IgM and surface IgD.
And these cells have left the bone marrow and travel
to the secondary lymphoid tissue. And they sit and
wait in the secondary lymphoid tissue in case their
particular antigen, the antigen they recognize