Immunology sheet # 6 - Mohammad Okdeh

Antigen (B Cells) specificity
And T Cells
Today our lecture will be, as the title implies, about antigens and their specificities and how does each component of the antibody contribute to this diversity. And we will talk about the maturation process of B cells. Also T-cells will be discussed briefly at the end of the lecture.
A brief summary here would be helpful, B cell development occurs through several stages, each stage representing a change in the genome content at the antibody loci. An antibody is composed of two identical light (L) and two identical heavy (H) chains, and the genes specifying them are found in the 'V' (Variable) region and the 'C' (Constant) region. In the heavy-chain 'V' region there are three segments; V, D and J, which recombine randomly, in a process called VDJ recombination, to produce a unique variable domain in the immunoglobulin of each individual B cell. Similar rearrangements occur for light-chain 'V' region except there are only two segments involved; V and J.
This random recombination generates about 1000 million different antibodies that can recognize 1000 million different epitopes of foreign antigens. (Wikipedia claims that it can generate up to about 10 billion antibodies rather than a billion).
The region (locus) of a chromosome that encodes for an antibody is large and it contains many distinct genes for each domain of the antibody, the locus containing heavy chain genes is found on chromosome 14, and the loci containing kappa and lambda light chain genes are found on chromosomes 2 and 22, respectively.
During the development of B cells, the first chain to be produced or rearranged is the heavy chain. But actually we are going to talk about the light chain first so things become easier.


- LIGHT CHAIN:
This type as we mentioned include both the kappa light chain and the lambda light chain. Again we will talk about kappa at first as it is easier.
- The kappa gene locus is divided into different gene segments, and if you examine it carefully, you’ll find that this chain initially has variable segments and they are referred to as V segments; there are about 100 “V” kappa light chain genes.
(The dr. said so, but upon searching, it appears that there are 35 genes for kappa and 30 genes for lambda light chains).
The other area that you’ll find is the J segment (J for junction or joining), an area with smaller gene segments, there are five “J” kappa light chain genes. After that we’ll have one C locus which is the constant segment.
Now from what was mentioned above and what we have already studied we know that light chain is composed of 2 domains: variable and constant domains.
Each domain codes for 110 amino acids, the constant domain is made of one segment then the C segment codes for 110 amino acids. But V segment of the Variable domain codes for about 100 amino acids and J-segments for the rest.
During the development, embryonic germ cells randomly recombine the components of the variable domain, for example it combines V1 with J4 or V37 with J2…..etc. This randomized process will actually give all possible combinations where every single difference gives different specificity. For the sake of our lecture we will assume that we have about 100 different V segments and 5 different J segments, this mathematically gives 500 different combinations.
But how does the process of recombination occur? Or what perform all the cleavage and rejoining processes that take place?
- Recombination Activating Gene (RAG) is a gene that produces all the enzymes that are responsible for this complicated process of separation, shuffling and rejoining.
After recombination take place segments other than those have been chosen are deleted and disposed off and this make the process irreversible and this also what makes the amount of DNA in mature B cell is less than that of the germ cell.
Now the constant domain is attached then an mRNA molecule is produced. Then the corresponding peptide, which is actually the kappa light chain, is produced.

- Lambda light chain is generally similar to kappa one. The major difference between the two is that each J segment in lambda has its own constant segment and this make the recombination much more complicated. Though, still the constant segment has nothing to do with the specificity issue.
Here somebody asked a question about the CDRs (Complementarity Determining Regions) and their role in the whole process of specificity?
CDRs are antibody or T cell receptor regions where the molecule complements an antigen's conformation. Thus, CDRs determine the molecule's specificity and make contact with a specific antigen. CDR1 and CDR2 are found in the variable (V) domain, and CDR3 includes some of V, all of diverse (D) (found in heavy chains only) and joint (J), and some of the constant (C) domains. CDR3 is the most variable.

- HEAVY CHAIN:
The variable domain in the heavy chain is composed of three joined segments rather than two: V segment, J segment and D segment (D for diversity). This new segment adds for the diversity of the antibodies. Here we have approximately 100 “V” heavy chain genes, about 12 “D” heavy chain genes and 4 “J” heavy chain genes.
In the developing B cell, the first recombination event to occur is between one D and one J gene segment of the heavy chain. Any DNA between these two genes is deleted. This D-J recombination is followed by the joining of one V gene, from a region upstream of the newly formed DJ complex, forming a rearranged VDJ gene. All other genes between V and D segments of the new VDJ gene are now deleted from the cell’s genome.
About 90 - 95 amino acids of the heavy chain variable domain come from the V segments and the rest of them come from the D and the J chain collectively.
Again as we can calculate the estimated count of recombination possibilities by multiplying the count of V, D, and J segments with each other, and by that we get about ~6000 different possibilities.
Multiplying 6000 we get here by 500 we get from the light chain variation, we will end up with 3 million possible specificities.
- B cells maturation:
Before we start talking about the process we should know that only ~10% of B cells produced in the bone marrow become fully mature B cells, the remaining 90% are cells that failed to proceed through maturation steps correctly for any reason, and that’s why this process is very wasteful and very faulty. Another thing to highlight is that, as we know, each one of us has 2 copies of each locus of his genetic material: one from his father and the other being from his mother, and here the cell can initiate the maturation process with either of them, equally.

Step 1 – heavy chain rearrangement:
- Heavy chain rearrangement begins first. Let’s assume using the paternal chromosome. If the rearrangement was successful, then the process proceeds. If it wasn’t, then maternal chromosome rearrangement should take place. Again if the results were in the right direction, the process continues. Otherwise the cell undergoes apoptosis.
During this stage, once we have a successfully arranged μ chain, it’s going to be expressed in the cytoplasm of B cell and this B cell will be known as pre-B cell.
Step 2 – light chain rearrangement:
- As mentioned above, two light chain types are present; Kappa and lambda light chains. The process of rearrangement theoretically can be started with either of the two, but strikingly it was found that kappa chain is preferred over lambda chain.
The same cycle that begins by choosing the parental chromosome to start with and ends up either by apoptosis or successful rearrangement, take place maximally twice, success in any step at this stage will produce the desired antibody:
The rearrangement begins with kappa chain of either source (paternal or maternal), successful rearrangement, gives the antibody while failure, moves the process to the next available option which is the chromosome of the other source. Again, success gives an antibody and failure moves the process to the next option which is the lambda light chain that has steps similar to those mentioned above.
Because of this preference, the total kappa to lambda ratio is roughly 2:1 in serum (measuring intact whole antibodies) or 1:1.5 if measuring free light chains.
- Now, you may ask yourself; although we said that there is 1000 million different specificities all we get is only 3 million (0.003 of what is supposed to be produced)!!! So where is the rest?!
- It appears that many other mechanisms are responsible for the generation of that number of different combinations; few of these additional mechanisms are the junctional diversity and mutations.
Junctional diversity:
To make things clearer I searched for this topic on Wikipedia and this is what I found, by the way it’s the same as what we should know in a comprehensive brief way:
Junctional diversity concludes the process of somatic recombination or V(D)J recombination, during which the different variable gene segments (those segments involved in antigen recognition) of TCRs and immunoglobulins are rearranged and unused segments removed. This introduces double-strand breaks between the required segments. These ends form hairpin loops and must be joined together to form a single strand (summarized in diagram, right). This joining is a very inaccurate process which results in the variable addition or subtraction of nucleotides and thus generates junctional diversity.
(The link : http://en.wikipedia.org/wiki/Junctional_diversity)
CONSTANT REGIONS OF THE HEAVY CHAIN:
- Heavy chains have five constant segments arranged as follows ; μ, γ, δ, ε and α. Each one of those segments codes either for 3 (γ, δ and α) or 4 (μ and ε) constant domains, depending on the type of the antibody.
As(μ), the segment coding for IgM, is the first C segment in the arrangement, it’s the first one to be encountered by the heavy chain after a successful rearrangement is over, and its product (IgM) will be the first immunoglobulin to appear on B cell surface, which is now called “immature B cell”. The next C segment will be the (γ), which codes for (IgD). When IgD is expressed on the surface of the immature B cell this will produce what is so called a mature B cell, in which both IgM and IgD appears on its surface at the same time. Recall that the two immunoglobulins have the same specificity. You never have different specificities on the same B cell!
Class switching regions:
Naïve mature B cells produce both IgM and IgD, which are the first two heavy chain segments in the immunoglobulin locus. After activation by antigen, these B cells proliferate. If these activated B cells encounter specific signaling molecules via their CD40 and cytokine receptors (both modulated by T helper cells), they undergo antibody class switching to produce IgG, IgA or IgE antibodies. During class switching, the constant region of the immunoglobulin heavy chain changes but the variable regions, and therefore antigenic specificity, stay the same. This allows different daughter cells from the same activated B cell to produce antibodies of different isotypes or subtypes (e.g. IgG1, IgG2 etc.). This switching is irreversible, you can’t convert the cell back to naïve cell after switching.
Since the variable region does not change, class switching does not affect antigen specificity. Instead, the antibody retains affinity for the same antigens, but can interact with different effector molecules.


T CELL RECEPTOR (TCR):
The T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells) that is, in general, responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules
The TCR is composed of two different protein chains (that is, it is a heterodimer). In 95% of T cells, this consists of an alpha (α) and beta (β) chain, whereas in 5% of T cells this consists of gamma and delta (γ/δ) chains. Here we are concerned with the alpha beta chains.
The beta actually replaces the heavy chain while the alpha replaces the light chain. So during the development of the (TCR) the first chain to be rearranged will be the beta chain, the variable domain of which has the same segments of that of the heavy chain has (V, J and D). The constant segment is composed of only one segment rather than five.
Alpha chain variable domain is produced by the VJ combination like that of the light chain, with one available constant segment only.
Diversity is more or less similar to that of B cell, because the specificity again is a function of the variable domain.
Note :
Receptors are actually present on the cell surface in both B and T cells, but what they should do is to affect the cell function, and in order to do so there are some structures that help in performing this. The receptor is not alone.
BCR: has alpha and beta chain that transmit messages to the intracellular matrix.
TCR : has CD3, zeta and eta that couples the receptor with intracellular pathways.
That’s all for today



God Bless You
Done By: Mohammed A. Okdeh
LEC #6 on 12/12/2010