VDJ rearrangement

Definitions & Key takeaways

VDJ rearrangement is a process by which the genes responsible for encoding the variable region of the B-cell receptor and T-cell receptor are rearranged to create a diverse repertoire of receptors capable of recognizing a wide variety of antigens.

During VDJ rearrangement, DNA segments called V (variable), D (diversity), and J (joining) are rearranged to form the coding sequence for the receptor variable region. The process is mediated by recombination-activating genes (RAG1 and RAG2) and involves the introduction of double-strand breaks at specific recombination signal sequences (RSS) flanking the V, D, and J segments, followed by joining of the broken ends.

The resulting receptor variable region diversity is crucial for adaptive immunity, allowing the immune system to recognize and respond to a wide range of potential pathogens. However, errors in VDJ rearrangement can lead to autoimmune disease, immunodeficiency, or cancer.

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The adaptive immune response is highly specific for each invader, and that’s because the cells of the adaptive immune response have receptors that differentiate friendly bacteria and potentially deadly pathogens from their unique parts - called antigens.
The key cells of the adaptive immune response are the lymphocytes- the B and T cells. And the antigen receptors for T cells and B cells have a lot of things in common, one of which is that they share the same mechanism - called VDJ rearrangement - to generate a massively diverse set of receptors.
VDJ stands for variability, diversity, and joining, respectively, and VDJ rearrangement has 4 key characteristics that help ensure that each antigen receptor is unique.
First, each individual inherits multiple V, D, and J gene segments; second, the V, D, and J gene segments randomly recombine - meaning that any V can pair with any D and any J; third, there’s recombinational inaccuracy - meaning that this process is purposefully error prone - which leads to additional variation; and fourth, there’s random reassortment of two chains - meaning that this process involves two different chains that come together to make the receptor.
First, let’s look at our antigen receptors. The B cell receptor, or BCR, is essentially an antibody, except that it has a transmembrane part that goes through the membrane and attaches the receptor to the surface of a B cell.
Just like antibodies, the B cell receptor has a heavy chain and a light chain. One region or fragment of the B cell receptor binds the antigen and it’s called the fragment - antigen binding or Fab region.
There are two Fab regions on every B cell receptor. Then there’s the constant region or fragment called the fragment - constant or Fc region, which is the part that determines what class of B cell receptor it will be - for example, IgM or IgD, and whether or not it will remain a membrane bound B cell receptor or if it will get secreted as a free floating antibody.
The T cell receptor, or TCR, on the other hand looks quite different but still only has two chains. In this case it has an alpha chain which is analogous to the B cell light chain, and a beta chain which is analogous to the B cell heavy chain.
Unlike the B cell receptor, the alpha and beta chains of the T cell receptor only form a transmembrane receptor - they don’t get secreted and there aren’t different classes.
They also only have one antigen binding site. So VDJ rearrangement, really only affects the portion of the protein that comes into contact with the antigen.
For both the BCR and TCR this is known as the variable region or V region.This region is supported by a constant or C-region.
The variable regions of each of these receptors is highly diverse across the entire cell population -where no two T cells or B cells developed will recognize the same exact antigen.
This is accomplished by the significant variability in the genetic sequence that makes up the V region - but this variability is concentrated to certain segments of the V-region known as hypervariable regions or complementarity-determining regions.
These locations are brought together when the two chains of either the TCR or BCR are brought together to create one hypervariable site at the tip of each arm of the BCR or the tip of the TCR which is the antigen binding site.
So VDJ rearrangement is used to create the variability that is seen in these hypervariable regions which form the actual antigen binding site, and only that part of the protein.
The first step in VDJ rearrangement happens before we are born, in the sense that we inherit from each parent, multiple V, D, and J gene segments.
Each person has 44 V gene segments, 27 D segments, and 6 J segments and that is just for B cell heavy chains, there are more V, D, and J segments for the T cell receptor beta chain and of course there are more V and J segments for the B cell light chain and T cell alpha chain.
The various gene segments are all lined up on the DNA like box cars on a train. The V gene segments are located furthest away from the genes that encode the constant gene segments, while the J segments are located fairly close to the constant gene segments.
And on the chromosomes that contain the genes for the heavy chain or beta chain the D gene segments sit between the V and J gene segments.To generate diversity, these gene segments reassort randomly in both B cells and T cells.
A bit like how you might have several pairs of shoes, pants, and shirts and can mix and match them to create lots of different outfit combinations.
For B cell we’ll start with the heavy chain and for T cells we’ll start with the beta chain. So let’s say for this heavy chain or beta chain rearranged to have V2-D2-J1, well this would be completely different and bind different antigens than a heavy chain or beta chain that rearranged to have V2-D3-J1.
So just by using a different D region, it completely changes what antigen these two cells might bind. The rearrangement of these different exons is accomplished by having recombination signal sequences which are noncoding DNA sequences that flank the points where DNA needs to be cut and rearranged.
A recombination signal sequence has three parts to it. First, there’s a 7 nucleotide sequence called a heptamer - 5’-CACAGTG-3’.
Second, there’s a stretch of either exactly 12 nucleotides, corresponding to 1 turn of the DNA double helix, or exactly 23 nucleotides, corresponding to 2 turns of the DNA double helix.
Third, there’s a 9 nucleotide sequence called a nonamer sequence - 5’-ACAAAAACC-3’. During VDJ rearrangement, the DNA moves together or loops around to bring together two recombination signal sequences depending on how they are oriented, so that they are both mirror images of each other in terms of their 5’ to 3’ orientation.
For example, let’s say that there’s a recombination signal sequence that flanks a V exon and another one that flanks a D exon.
A group of enzymes called RAG1 and RAG2, which are encoded by recombination activating genes cut the DNA at these recombination signal sequences.
Then repair proteins like Ku, DNA-protein kinase, and Artemis help to reattach and therefore recombine the DNA, which results in an extrachromosomal circular piece of DNA when it is cut out which gets lost when the cell divides.
In this way, each daughter cell will have the exact same V-D-J sequence as the original cell that recombined the gene segments during VDJ rearrangement.
At this point we are going to increase diversity even more, because this process is inherently error prone! The cut end is going to be added onto by the enzyme terminal deoxynucleotide transferase or TdT which randomly adds and removes nucleotides.
These are called N-nucleotides and they can literally be any-nucleotide. So there could be two cells that use the exact same V, D, and J regions to create their heavy or beta chain, for example V2-D3-J1, but they might differ because of recombinational inaccuracy and the enzyme TdT.
So really one cell with that V2-D3-J1 selection might be V2-Adenine-Thymine-D3-Cytosine-Guanine-J1 where adenine, thymine, guanine, and cytosine are added in by TdT and the other cell would be V2-Cytosine-D3-Guanine-Adenine-J1.
And these additional changes alter the antigen specificity of the receptor. Finally, the DNA ends are held tightly together and are then joined by a complex of enzymes which include DNA ligase IV which ligates the DNA together again creating the new coding joint with the newly rearranged V, D, and J regions.
These cuts and processes will need to happen to join D to J and then V to D-J. At this point we’ve made a heavy chain for a B cell or a beta chain for a T cell.
Before we proceed with making the light chain for the B cell or an alpha chain for a T cell, it’s important to test if the chains we made can bind normally to light or alpha chains.
To check that in B cells, the heavy chain is paired with a surrogate light chain known as VpreB, and in T cells, the beta chain is paired with a pre-T alpha surrogate chain.
These surrogate chains, VpreB and pre-T alphas are like training wheels for the heavy chain or beta chain. If the heavy chain or beta chain is able to bind to the surrogate receptor, it signals the nucleus of the cell to wildly proliferate forming lots of daughter cells.
Each daughter cell then goes through a process called a random assortment of chains, which is where it creates a light chain or alpha chain to pair with its heavy chain or beta chain.
The light chain or alpha chain then goes through V-J rearrangement as we did for the heavy or beta chains randomly choosing a V and J region to put together.
Remember, there are no D regions in light or alpha chains. Once it has rearranged its light or alpha chain it will pair with the already made heavy or beta chain.
If the new light chain or alpha chain binds to the heavy chain, then the cell is tested to make sure that it’s not self-reactive.
If the cell is self-reactive, it will keep trying to rearrange the light or alpha chain until it runs out of genetic material.
And if it does run out of genetic material, then the cell dies. Alright, as a quick recap, the goal of VDJ rearrangement is to create a massive amount of unique antigen receptors on T and B cells.
During VDJ rearrangement the cell randomly selects from the multiple V, D, and J regions we inherit. Cuts are made in the DNA sequence at precise locations that are signaled by the Recombination Signal Sequence which is made of a heptamer, nonamer, and either a 12 or 23 base pair spacer.
The cuts are filled in with the error prone enzyme TdT which will add N-nucleotides at joining sites and then the DNA is ligated together.
Once the cell has completed rearranging a beta chain or heavy chain it rapidly proliferates, and that chain is paired with a wide variety of alpha or light chains.