1 Introduction
The key to a properly functioning immune system is its ability to reliably distinguish between its own and alien elements. The nonspecific response of the immune system involves the recognition of the characteristic parts of invading pathogens and forms the body’s first line of defence against infection. The constantly evolving relationship between the host and the pathogen and the resulting on-going change in the structure of pathogens that could be
2 Recombination of the Genes of T and B Cell Receptors
The cells that perform a key role in the functioning of the adaptive immune system are T and B lymphocytes. Generally speaking, T and B cells are responsible for the body’s second line of defence. They respond precisely to particular antigens by producing antibodies, creating substances that activate other types of cells to combat the invading pathogens and directly killing virus-infected cells and tumorous cells. An important feature of the adaptive immune system is its immunological memory, which enables a quick response once the body comes in repeated contact with a given antigen. B and T cells produce antibodies and TCR molecules, respectively, i.e. antigen receptors (Figure 3.1). Both types of cells consist of the constant domain, which anchors the receptor in the cellular membrane, interacts with other molecules and transmits signals into the cell, and the variable domain, which is responsible for the specific recognition of antigens. A key characteristic of T and B cells is that each cell produces only one type of receptor for a specific antigen. Therefore, the diversity of antigen receptors is due to a large number of lymphocytes, each with a unique receptor.
The variable parts of protein chains in antibodies (Ig) and TCR molecules are coded in the genome of a foetus by noncontinuous gene segments which appear in many variants, called V, D and J minigenes. Genes encoding the
Structure of a T cell receptor (based on TCRαβ) and an antibody
V(D)J recombination (Figure 3.2) can be divided into two phases: the DNA cleavage phase and the DNA repair phase. In the first phase, the RAG-1 and RAG-2 proteins (McBlane et al., 1995) recognise signal sequences and form a
To better understand the extraordinary efficiency of V(D)J recombination in generating the diversity of genes that encode antigen receptors, let us briefly analyse the case of genes coding TCRαβ. The α chain of this receptor is coded by the V and J minigenes. The two minigenes occur in the genome in ~45 and ~55 variants, respectively. Thus, about 2,475 (45 × 55) different TCRα genes can theoretically be created. The TCRβ chain is coded by the V, D and J minigenes occurring in ~50, 2 and 12 variants, respectively, which means that 1,200 different β chains can be created. Therefore, the combinatorial diversity alone allows for the creation of about 3 × 106 different TCRαβs (2,475 TCRα × 1,200 TCRβ), i.e. about 100 times more than all other genes put together. The addition of Ps and Ns to the coding ends is part of the mechanism of the aforementioned joint diversity, which, along with combinatorial diversity, allows for the creation of about 1015 different TCRαβs – 10,000 more than the number of stars in our galaxy.
3
The method for obtaining a wide variety of receptors has a significant drawback: the same mechanism that allows for the random joining of minigenes and the addition of random nucleotides also creates the risk of producing nonfunctional receptors or, even worse, receptors that target the body’s own antigens (autoantigens), leading to autoimmunity. To prevent the formation of cells with dysfunctional or autoaggressive receptors, the maturation of T and B cells uses selection mechanisms. Let us analyse the development of lymphocytes based on T cells of the αβ line (Figure 3.3). At the stage of DN2 and DN3 cells (with the phenotypes CD4-8-CD25+CD44+ and CD4-8-CD25+CD44-) during the development of Tαβ cells in the thymus, the locus coding TCRβ undergoes recombination: the Dβ and Jβ minigenes are joined together first, followed by the Vβ and DβJβ minigenes (Cobb et al., 2006; Krangel, 2003). If the recombination is successful, the newly-created TCRβ chain is expressed and, along with the pre-Tα chain (with which it creates a pre-TCR molecule) (Groettrup et al., 1993), sends a signal that saves cells from death and causes their rapid proliferation. This allows for the selection of cells in which the gene coding TCRβ was rearranged correctly and encodes a functional protein, i.e. a protein that can bind with pre-Tα, can be exported onto the surface of the cell and is able to send a signal inside the cell. The signal from the pre-TCR also shuts down recombination, preventing the recombination of the minigenes of the other allele and ensuring that only one β chain of TCR is produced in the cell (Löffert et al., 1996). The recombination of the TCRα locus, during which the Vα and Jα minigenes are joined together, takes place in double-positive cells (CD4+CD8+). The TCRαβ molecule can already be created at this stage, and the cells where the molecule is expressed undergo selection to test for the potential usefulness or harmfulness of the receptor. During the selection, autoantigens are presented to lymphocytes in the context of an MHC molecule, i.e., the same process is as it is later when non-self antigens are recognised during the immune system’s normal response. A lack of interaction between TCR and the MHC/autoantigen complex blocks the death-preventing signal from entering the lymphocyte, which dies as a result (Kisielow, Teh, et al., 1988). Thus, lymphocytes with a useless receptor, i.e. one that is unable to recognise any antigen in the context of the MHC molecule or transmit the signal, are eliminated. A very strong signal resulting from a strong interaction between the lymphocyte and the presenting cell also leads to the death of the lymphocyte as it indicates that the receptor has recognised the autoantigen.1 About 2% of lymphocytes survive both types of selection (positive and negative), i.e. those that, following
Maturation of Tαβ cells in the thymus and the selection of the TCR repertoire
Another issue with V(D)J recombination is the potential harm of the process itself. Compromised DNA integrity during recombination, which involves cutting and combining DNA, carries the risk of errors that can lead to DNA translocation and neoplastic transformations. Thus, V(D)J recombination is controlled by many factors: the availability of DNA for RAG-1/RAG-2 recombinase, tissue specificity, timing, the number of cells involved, and, most importantly, precise control over the expression and activity of RAG-1 and RAG-2. The final section of this paper will discuss the evolutionary origin of RAG-1 and RAG-2 genes and the hypotheses explaining the mechanisms controlling their expression.
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The adaptive immune system that takes advantage of T and B cells, which show a vast diversity of antigen-recognising receptors created during V(D)J recombination, is unique to jawed vertebrates (Gnathostomata). This means that all key elements of the system must have evolved between 450 and 500 million years ago (between the divergence period of the Agnatha and the Chondrichthyes). Researchers who have attempted to find the reason for the immunological “big bang”, as it is referred to, have proposed the hypothesis that RAG-1 and RAG-2, i.e., the genes which code the enzymes required for recombination, entered the genome of a common ancestor of jawed vertebrates through a horizontal transfer as a mobile genetic element, i.e. a transposon (Schatz, 2004). The hypothesis is supported by the fact that in biochemical terms, V(D)J recombination resembles transposition, the signal sequences (which flank the minigenes) resemble the sequences that flank transposons (Cowell et al., 2004) and the RAG-1 and RAG-2 proteins themselves are able to perform a transposition, i.e. to cut away a DNA fragment and insert it somewhere else in the genome (in vitro, but also, though very rarely, in vivo) (Agrawal et al., 1998; Hiom et al., 1998; Messier et al., 2003). Furthermore, RAG-1 and RAG-2 genes are located directly next to each other, and their protein-coding segments are enclosed within single exons (Oettinger et al., 1990). This compact structure resembles that of transposases (enzymes coded by transposons). The hypothesis concerning the transposon origin of RAG recombinase has been reinforced by the discovery of transposases from the Transib family in many species of invertebrates whose structures resemble fragments of the RAG-1 protein (Kapitonov & Jurka, 2005). Thus, the evolution of the whole system would be as follows (Figure 3.4): a transposon coding RAG transposase was integrated with the genome of a common ancestor of jawed vertebrates, and a DNA fragment that contained only signal sequences and whose mobility likely still depended on the enzymatic activity of transposase was created.2 The transposition of such a fragment into the coding part of the pre-receptor gene would make the expression of a functional version of the receptor fully dependent on the activity of RAG transposase, which in turn would be the only enzyme able to restore the continuity of the open reading frame. A series of duplications and mutations of the pre-receptor gene would lead to the creation of the minigenes and all loci coding the antigen receptors known today. The integration of the RAG transposon, in addition to the potential benefit of diversifying antigen recognition receptors, must have enforced the evolution of mechanisms for preventing threats to genome stability arising from the activity of transposase.
The scenario of the transposon hypothesis for the origin of RAG-1 and RAG-2
Let us, however, go back to the very beginning when the RAG transposon was introduced into the genome of a common ancestor of jawed vertebrates. One should bear in mind that genomes are quite literally “littered” with the remains of many transposons that, throughout evolution, “infected” the genetic material of their hosts. The integration of transposons with the host genome is (from the host’s point of view) harmful, primarily due to the danger caused by the uncontrolled ability of the transposon to move within the genome, potentially damaging the host’s genes. Consequently, organisms have evolved defense mechanisms that help hamper the activity of transposons, mainly by silencing their expression through epigenetic changes in DNA structure (DNA methylation and/or histone modifications). How, then, can the fact that the transposon containing the RAG-1 and RAG-2 pre-genes was not silenced be explained? Analysis of the few cases where genes originating from transposons
Characteristic locations of genes originating from transposons that still function in their hosts’ genomes. Blue rectangles mark the exons of the host’s gene, green ones mark its promoter and red ones mark transposase genes. Arrows indicate the direction of transcription
One such location is a DNA fragment located directly downstream of an existing gene. In such a case, later changes create a hybrid gene coding a protein with a modified function (compared to that of transposase and the original host protein). Another characteristic location is the head-to-head arrangement, whereby the transposon integrates itself above an existing gene and the directions of the host gene and transposase expression are opposite (Kalitsis & Saffery, 2009). It has been shown that in such cases, the host’s genes have a constitutionally active two-directional promoter that may trigger the expression of both the host’s gene and the sequences located above it. From the viewpoint of the transposon, the second option is more beneficial, as after integrating with the genome, the transposase gene will have a guaranteed mechanism of expression and any attempts at silencing it would also hamper the expression of the host’s gene. Furthermore, the structure of the host’s gene and the transposase gene is left intact (in contrast to the first option), which allows both genes to perform their original functions. There are very good reasons to conclude that the integration of the RAG transposon involved the second of the aforementioned mechanisms and the properties of the host’s NWC gene enabled the integration and survival of the RAG transposon.
The NWC gene (Cebrat et al., 2005) is the third gene in the locus containing RAG-1 and RAG-2. The location of the locus is conserved in all species of jawed vertebrates (Figure 3.6). The orthologs of the NWC gene are also present
Structure of the RAG/NWC locus. The green rectangles mark the exons of the NWC genes, the blue rectangles mark RAG-2 and the red rectangles mark RAG-1. Horizontal arrows show the directions and range of transcription. Vertical arrows show the effect of cis-regulatory elements (yellow ovals) on the promoters of RAG-1 and RAG-2
As it has been stated in previous sections of this chapter, the expression of RAG-1 and RAG-2 falls under very precise control: it is well-coordinated and takes part in specific moments of T and B cell development. The elements responsible for this control are cis-regulatory elements. These are sequences located predominantly on the 5’ side of the RAG-2 gene which, once the appropriate transcription factors are attached, interact with RAG-1 and RAG-2 promoters, leading to their activation in the maturing lymphocytes (Figure 3.6) (Hsu et al., 2003; Wei et al., 2002; Yannoutsos et al., 2004; Yu et al., 1999). The control elements for the expression of RAG s (both the cis-regulatory elements and the promoters) are considered a later evolutionary acquisition. The promoter of the NWC gene, in contrast to the RAG s, is active in non-lymphoid cells (Cebrat et al., 2005, 2008). The constitutive activity of the NWC promoter originated from its other properties which, as mentioned above, were found to have been key during the first stages of coexistence between the RAG transposon and the genome of the common ancestor of jawed vertebrates. However, this activity could have been harmful to the correct functioning of the control mechanisms for the expression of RAG s during lymphocyte maturation. This may stem from the fact that the transcription of the NWC gene, involving the area with cis-regulatory elements that activate RAG promoters, could interfere with the binding
It is fascinating that the activity of what in essence constitutes a molecular pathogen, i.e., the RAG transposon, ultimately led to the development of a seemingly simple, yet complex system that is very effective at combating other pathogens and determines the body’s molecular “self-awareness”. However, one should remember that the evolution of this system also depended on the interaction between the RAG transposon and the host’s genome. A complete characterisation of the mutual relationships in the control mechanisms for the expression of RAG and NWC genes, an investigation into the control mechanisms for the expression of NWC genes in invertebrates, and the characterization of the NWC protein function should improve our future understanding of the evolutionary processes that have led to the emergence of the adaptive immune system.
Notes
Some of the lymphocytes that survive the strong signal resulting from the recognition of the autoantigen constitute the exceptional CD4+8- subpopulation of T cells, called regulatory lymphocytes (Wojciech et al., 2014), which are later used to control the action of conventional lymphocytes, e.g. when the immune response is silenced, and which constitute the second line of defence (after the selection in the thymus) against autoaggresive lymphocytes.
Perhaps this occurred as a result of the duplication of the RAG transposon and the incomplete transposition of the duplicate into another location in the genome.
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