VDJ recombination - Grade: 2.1 PDF

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Summary

Exploring what VDJ recombination is and how important it is for an effective immune system. Detailing the process and the steps involved....

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Justify the following statement “V(D)J recombination is crucial for the effective functioning of the immune system” Our adaptive immune system is essential in the defence against infection, it is a slow process usually taking days but it provides long-lasting protection from any encountered pathogens. In order to fulfil its immune role, the adaptive system needs to be able to identify and destroy foreign antigens, to do this it recruits two major cell types - B cells and T cells. These lymphocytes possess a wide range of surface receptors which they use to recognize an infinite amount of pathogens, a feature which has been described as the “hallmark” of the adaptive system. The way in which we are able to generate such diversity has been a question for many years, sparking up theories such as The Germline theory which states that one gene makes one antibody; such an idea would mean that our genome would be entirely devoted to just lymphocyte receptors, therefore, making the theory impossible. Another theory referred to as the Somatic-variation theory was also proposed, this theory focuses more on antibody diversity, claiming that a small number of immunoglobulin genes are able to generate a large number of antibody specificities from recombination in mutation. Yet again this theory can not be true as some regions remain constant and it does not take into account class switching. In 1976, work by Tonegawa demonstrated that during B cell differentiation, the separate gene segments encoding the variable and constant regions were rearranged. This evidence introduced the idea of VDJ recombination, a process that involves the recombining of gene segments within the antibodies and the T cell receptors to generate a much-needed amount of diversity for antigen recognition. Furthermore, it provided evidence of how the immune system has evolved and developed a way in which to generate a vast number of receptors from a small number of genes, in fact, humans are able to synthesise from 1010-1011 antibodies from only 21,000 genes. Mechanism VDJ recombination is a perfectly coordinated cascade of reactions that occur in the primary lymphoid organs, more specifically the bone marrow for B cells and within the thymus for the T cells during the early stages of their maturation. In the recombination process, two different antigen recognition sites are paired together, for the antibodies this is the heavy and light chain, with the V region acting as the antigen-binding site and providing specificity. Whereas for the T cells, their recognition site aka their receptor is comprised of a heterodimer of the two transmembrane polypeptide chains, alpha and beta, covalently joined by disulphide bonds. These alpha and beta chains are able to contribute to the recognition specificity for the presented peptide on the appropriate MHC complexes. Within the chains of these recognition sites are the all-important gene segments, variable (V), diversity (D) and joining (J), the heavy and Beta chains contain all three segments whereas the light chain and alpha chain consist only of V and J.The Recombination Activating Genes (RAG1 and RAG2) are the orchestrators of the recombination process. The process begins when RAG1 and RAG2 recognise the Recombination signal sequences (RSSs) associated with the V(D)J segments, the RSSs signal for RAG1 and RAG2 to complete the recombination process and join D-J and V-J in the heavy chain and V-J in the light chain. The RSSs flank each germ-line V, D and J gene segment, more specifically the 3’

end of each V gene, 5’ end of each J gene and both sides of each D gene segment (Figure 1). Following the recognition of the RSSs by the recombinases, the gene segments are brought together by the bending of DNA in either deletional joining or inversional joining, creating a structure called a synapse. Then at the two junctions of the coding sequences and RSSs, the RAG1/2 complex cleaves one of the DNA strands, resulting in a single-stranded break. It’s important to mention that these events occur simultaneously on both gene segments. The free 3’OH group from the cut single strand performs a nucleophilic attack on the phosphodiester bond, linking the opposite strand to the signal sequence and therefore creating a hairpin loop as well as a double-stranded break. The hairpin is then cut, generating sites for the addition of P nucleotides to fill the overhang; addition of P nucleotides also increases the variability. For light chains, this where the process finishes and the gene segments come together, however for the heavy chains there is still one more step. In this final step, the enzyme terminal deoxynucleotidetransferase (Tdt) attaches N-region nucleotides at the cut ends of the V, D and J coding sequences. It is crucial that the additional nucleotides, both P and N region come in triplets in order to maintain the open reading frame of the gene and prevent the formation of what is known as a non-reproductive rearrangement. Finally DSBR enzymes mediate repair and ligation to join the gene segments together, completing assembly of the heavy chain. V(D)J recombination results in the formation of a coding joint ( found between the coding sequences) and signal join between the RSSs. The heavy chain comes together first, successful assembly then triggers the assembly of the light chain.

Figure 1: An illustration of the Recombination Signal Sequences flanking the VDJ gene segments of the light chains ( lambda and kappa) and the heavy chain. RSSs are situated at the 3’ end of each V

gene, the 5’ end of each J gene and on both sides of the D gene segment. This signal allows for recombination by the recombinases.

Contribution to the immune system T lymphocytes are a key component of the adaptive immune system, they can be differentiated into either T Cytotoxic cells or T Helper cells, with the latter being regarded as one of the most important cells in adaptive immunity as it is essential for almost all of the responses. T cells are activated when their TCR recognises antigens presented to them by their corresponding Major Histocompatibility Complex (MHC), triggering responses that lead to the destruction of the pathogen. T Helper cells recognise antigens on MHC II, this along with costimulation activates the cells to secrete cytokines that stimulate the humoral and cell-mediated immune responses, furthermore, these helper cells also have an important role in the activation of B cells to secrete antibodies and Cytotoxic T Lymphocytes (CTLs) to kill target cells. These events all make for an effective immune system and are only possible because of the diversity generated to allow antigen recognition by the TCR. Antibodies have a very prominent role in adaptive immunity; these structures are required for the recognition of foreign antigens that have invaded the body. They bind to complementary epitopes, which are hydrophilic amino acids located on the surface of the antigen. As antibodies cannot kill pathogens directly by binding they must induce immunoglobulin-mediated effector functions which include: Antibody dependent cell mediated cytotoxicity (ADCC), activation of complement using either IgG or IgM, precipitation, neutralisation and agglutination. These methods allow for efficient removal of any encountered pathogens. The diversity generated by VDJ recombination through the recombinases is very essential because it allows the antibodies to recognise a multitude of antigens and therefore allows them to be removed immediately through the aforementioned effector functions. In summary, VDJ recombination and the diversity it generates are crucial for the effective functioning of the immune system as the adaptive response relies heavily on antigen recognition by the antibodies and the T cell receptors. An absence of recombination would result in a compromised immune system, consisting of an adaptive response with lymphocytes unable to respond fully or at all to any invading antigens due to a lack of recognition from limited diversity....