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ANTIBODIES AND T CELL RECEPTORS
|One of the characteristics of jawed vertebrates is the possession of adaptive immunity which allows them to predict a great diversity of potential antigens to defend against, attack antigens with specific humoral proteins, and retain a memory of the antigens they have encountered. In addition to the MHC and complement factors already considered, this system depends on two novel types of immunoglobulin known only in gnathostomes: antibodies and T cell receptors. These receptors are common in the human lymphocytes pictured below.|
|While these lymphocytes are the second most abundant type of leukocyte in the blood, the majority of them are found outside the blood in sites such as lymph nodes, the spleen, tonsils, the appendix, and Peyers patches of the small intestine. The following image is of the spleen.|
Lymphocytes can generate a diversity of 10,000,000,000,000,000 different receptors and each lymphocyte carries 105 copies of one receptor (Laird, 2000). Where did these complex proteins come from? Antibodies and T cell receptors are assembled from smaller chains, known as constant (C), variable (V), diversity (D), and joining regions (J). Some of these components evolved long before antibodies and T cell receptors. Variable immunoglobulin domains are known in cell surface molecules of plants and fungi in addition to a diversity of animals, including the most primitive animals, the sponges (Muller, 2001; Muller, 2001a). The V and C regions of immunoglobulins resulted from the duplication of an ancestral region of about 110 amino acids (Schluter, 1997). The ancestral Ig/TCR receptor probably included both a V domain and a C domain. The C1 domain of antibodies and TCRs is known in MHC molecules I and II, tapasin, and SIRPS (Du Pasquier, 2004).
The J chain of vertebrate antibodies is thought to function in the dimerization of antibody subunits and their transport. The gene for the J chain is expressed in a number of protostome invertebrates as well as in diverse groups of vertebrates. In invertebrates, it is expressed on macrophage-like blood cells and epithelial surfaces. Since these animals lack antibodies, it appears that the J chain functions in other capacities as well (Takahashi, 1996). The J chain is expressed one week before the mu chain in human fetal development, consistent with the suggestion that it serves multiple functions (Takahashi, 1996).
Vertebrates possess two types of immunoglobulin which are thought to be similar to the ancestral immunoglobulin which gave rise to antibodies and TCR genes, the JAM/CTX and the nectin/poliovirus receptor (PVR) families. These proteins serve as cell adhesion molecules and virus receptors (such as polio, Coxsackie virus, and reoviruses). Apparently, one ancestral pair of these genes was linked and subsequently duplicated to produce the four paralagous linkage groups known in the human genome. JAM, CTX, and nectin molecules can form dimers, as can the subunits of antibodies and T cell receptors (Du Pasquier, 2004). Drosophila possesses a protein with V and C chains showing homology to nectin (Du Pasquier, 2004). Tunicates, among the most primitive chordates, possess JAM, CTX and PVR genes which possess both a V and a C domain (Du Pasquier, 2004).
Jawless fish have no organized lymphatic tissue and lack both a spleen and thymus. Lampreys and hagfish seem to lack antibodies, T Cell receptors, MHC proteins, and RAG. (Zarkadis, 2001).
They do, however, possess serum heterodimeric proteins which resemble both antibodies and T cell receptors (Varner, 1991). These receptors are formed from two different heavy chains attached to two different light chains, unlike the antibodies and TCRs of vertebrates. While hagfish can reject foreign skin grafts, they do so with very modest increases in the amount of antibody-like moelcules in their blood (0.3% of serum protein compared to 50% in sharks) (Varner, 1991). Lampreys and hagfish do, however, possess lymphocyte receptors which are modified in development to produce a considerable diversity of proteins. They are not homologous to the antibodies and TCRs of jawed vertebrates and instead represent an independent mechanism for generating a diversity of leukocyte receptors (Pancer, 2005).A lamprey intestine is depicted below (with associated blood vessels).
Lamprey blood contains cells which are morphologically indistinguishable from mammalian lymphocytes although no MHC, T cell receptor, or antibodies are known from lampreys. These cells express genes associated with mammalian lymphocytes and occur in tissues (such as the intestines) where lymphocytes are common in higher vertebrates (Mayer, 2002). Lymphocyte-like cells in lampreys are small with little cytoplasm and produce lymphocyte transcription factors (Spi and Ikaros), CD45, BCAP, and CAST (which in mammals are primarily expressed in lymphocytes), CD98 and CD9 (which mammals use in lymphocyte proliferation and migration), proteasome subunits (PSMB4, PSMB7, 26S subunit pUb-R3, PSMA2, PSMA6, and PSMF1), and ABC9 (similar to the ABC proteins which mammals use to transport peptides to the MHC)., the complement protein C1q, and a number of other genes expressed in mammalian lymphocytes (hepsin, sygin 2, RAMP4, and talin) (Mayer, 2002).
In addition to antibodies such as IgM and IgW, sharks synthesize the antibody-like molecules IgNARC (new antigen receptor in cartilaginous fish) and NAR (new shark antigen receptor) (Schluter, 1997). The NAR immunoglobulin (new antigen receptor) known in nurse sharks functions as an independent molecule without forming a dimer with other immunoglobulins. Its variable region seems to be equally related to both antibody and TCR variable regions and may be derived from the ancestor of both (Roux, 1998). The variable regions of shark IgNAR regions have undergone elevated rates of mutation which affect antigen binding (Stanfield, 2007). Sequence comparisons suggest that NAR antibodies seem to have been derived from cellular adhesion molecules (rather than the Ig/TCR group) which were modified for immune function (Streltsov, 2004; Wilson, 1997) One set of fish proteins are referred to as NITRs or novel immune-type receptors. NITRs may be similar to the ancestral antigen receptor given that they possess a V, J, and C domain (Kasahara, 2004).
In sharks, B cells make 4 classes of antibody, including IgM, which humans also produce. IgD has been shown to be homologous to IgW and therefore this human antibody is a modified derivative of an ancestral antibody dating to the origin of jawed fish. IgD's expression in modern vertebrates is variable, suggesting that its role in immunity is less fixed than that of other antibodies (Ohta, 2006)
Shark antibodies can be secreted and can function on the cell membrane. While higher vertebrates create antibody diversity by possessing large numbers of antibody parts in gene clusters which will be randomly shuffled 50 V, 30 D, 6 J, 8 C), sharks possess more than 100 clusters which do not contain this diversity (1 V, 2 D, 1 J, 1 C) and are not reshuffled (Hohman, 1993). To express it another way, sharks possess (VH-D-D-JH-CH)n –an organization known as cluster-type--while bony fish and tetrapods possess (VH)n-(D)n-(JH)n-(CH)n—an organization known as translocon-type (Magor, 1999). Coelocanth antibody gene organization is part-way between the translocon and cluster-types (Stavnezer, 2004).
The antibody light chains in sharks and teleost fish form a number of clusters which share a V-J-C organization (Pitstrom, 2002). One type of immunoglobulin in sharks, IgW displays characteristics which might be expected from a primordial immunoglobulin (Berstein, 1996) In addition to IgM, lungfish possess immunoglobulins similar to the IgW previously known only in sharks. Thus, IgW and IgM must have duplicated early in the evolution of gnathostomes (Ota, 2003).
Unlike mammalian antibodies, the shark antibody
components are not reshuffled. Amphibians possess a receptor (CTX in Xenopus), which is similar to TCR and immunoglobulins but is formed by splicing two half-domain exons rather than by somatic rearrangement. It may represent a protein similar to the ancestral immunoglobulin prior to the establishment of somatic rearrangement (Chretien, 1998).Mammals
do however possess families of receptors such as paired Ig-like receptors (PIR), Ig-like
transcripts (ILT; also known as leukocyte inhibitory receptors LIR), and
killer inhibitory receptors (KIR) which can interact with MHC I and II
proteins. These antibody-like molecules do not rearrange
themselves (like antibodies and TCR receptors) yet they function in immune
responses (Laird, 2000). The KIR
gene family has undergone considerable expansion in apes since their separation
The adaptive immunity of higher vertebrates
depends on the ability of recombination activating genes (RAG) to randomly
join segments of antibody genes to create an enormous variety of antibodies
and T cell receptors. The RAG proteins
in vertebrate genomes seem to have resulted from the insertion of a bacterial
transposon of the Hin
recombinase family and the RAG sequence functions
in a way similar to transposons. When the RAG transposon
was inserted into the ancestral immunoglobulin gene, it seems to have
separated V and J segments which were once part of the same exon
RAG-1 regulates the recombination of B cell receptor regions κ, λ, and μ and the α, β,δ, and γ regions of T cell receptors (Schatz, 1989).
The RAG rearrangements only affect one type of leukocyte—it did not interrupt the preexisting innate responses (Du Pasquier, 2004). All gnathostomes can rearrange antibody and T-cell receptor segments using RAG genes. RAG I is homologous to integrase and RAG II is homologous to integration host factor; these two genes cause site specific recombination in bacteria (Bernstein, 1996).
Antibodies are produced
by B lymphocytes (present in tonsil pictured above). Antibodies can induce different responses: some
can be inflammatory, lytic, phagocytic
or allergic (
B-1 B cells produce autoantibodies without being stimulated by interaction with an antigen. This may represent a transitional stage between innate and acquired immunity mechanisms (Czompoly, 2006).
1) HEAVY CHAIN sequences/genes: 14q32
The heavy region of antibodies can interact with complement proteins, and with cell membrane receptors such as immunoglobulin receptors on epithelial cells and Fc receptors (Stavnezer, 2004). Heavy chains have constant, variable, diversity, and joining regions encoded by CH, VH, DH, and JH sequences. Some refer to each sequence as a separate gene (thus up to 300 genes , 4 of which are needed to produce a functional heavy chain) while others refer to this as one supergene (with up to 300 exons, 4 of which are needed to produce a functional heavy chain. On chromosome 14q32, the order of these sequences is:
5’---VH (some estimate more than 250 sequences)---JH (5 sequences)--- DH (at least 10 sequences) and CH (at least 10 sequences)---3’
This set of sequences stretch for 2.5 to 3 megabases (million DNA bases) (OMIM)
In sharks, IgNAR antibodies consist only of heavy chains and camels are known to make antibodies which consist only of heavy chains (Schaerlinger, 2008).
I) Variable Regions
There are a large number of variable sequences (estimates range from 100 to 250)
--These VH (variable heavy) sequences can be grouped into 7 classes based on the homology of the sequences. VH3 is the largest family with over half of the sequences. Interestingly, there are 2 other areas of the genome which contain VH genes—there are 2 sequences on chromosome 15 and 4 sequences on chromosome 16. Around 20 million years ago, these sequences arose from those located on chromosome 14 and are not functional (they lack joining sequences and, since interchromosomal recombination does not occur in the creation of antibody genes, they do not produce functional antibody sequences). The estimated number of VH sequences will undoubtedly change as the area is better studied: one study of one fraction of the VH region found 79 pseudogenes (twice the number of functional sequences in the area).
II) Joining Regions
--there are at least 5 sequences here (including one pseudogene) in an area about 8 kb in length.
III) Diversity Regions
--there are at least 10 sequences on chromosome 14 and one functionless sequence on chromosome 15.
IV) Constant Regions
--The heavy chain constant sequences are arranged in a 300 kilobase segment in the order: 5’---IgM—IgD---IgG3---IgG1---IgE pseudogene---IgA1---IgG2---IgG4---IgE---IgA2---3’
--The primordial cluster seems to have existed as IgM—IgD—IgG—IgE—IgA; after which the IgG sequence duplicated. Following the duplication of the IgG region, there was a duplication of the IgG—IgE—IGA regions resulting in 4 IgG regions and two regions each of IgE and IgA.
--IgM—There is no variation known in functional in the IgM constant region. Mutations here can cause one form of agammaglobulinemia. IgM antibodies are the main receptors on B cells and can be secreted as pentamers in which 5 separate antibodies are joined to a an IgJ polypeptide.
Cartilaginous fish and lungfish produce the antibody types IgM, IgNAR and IgW. Teleosts are known to produce five classes of antibodies: IgM, IgD, IgH, IgT and IgZ. In amphibians, IgM, IgD, IgY (a precursor to IgE and IgG), IgX, IgF, and a new class, IgP have been identified (Schaerlinger, 2008). IgD is homologous to IgW, an antibody class present in cartilaginous fish. It has been modified in different ways in diverse vertebrate lineages including cases in which it has been made a transmembrane antibody, a secreted antibody, and lost altogether (Ohta, 2006). Non-mammalian tetrapods possess IgY as the major secreted antibody and lack the related mammalian antibodies IgG and IgE. Reptiles are only known to express IgM and IgY. Some non-mammalian tetrapods use IgY to transfer passive immunity from females to their offspring (Belov, 2003; Stavnezer, 2004). It seems that there was a duplication of the IgY gene in the ancestors of mammals (prior to monotremes) and the two forms were modified to form IgG and IgE (Vernersson, 2004). All mammals use IgM, IgG, IgA, and IgE and lack the IgY used in reptiles (Belov, 2003). Monotremes have duplication of IgG genes but this was a separate duplication from that in humans and mice (Stavnezer, 2004). IgD is known in rodents, primates, artiodactyls and teleosts (Belov, 2003). Rabbits have 13 IgA regions (Stavnezer, 2004).
The human intestines are colonized by 500-1000 species of bacteria (Suzuki, 2007). At least 80% of the body’s plasma cells reside in the mucosal layer of the gut where they produce 40-60 mg of IgA per kg of body weight per day. (Thus, IgA is produced in greater quantities than any other class of antibody.) IgA can act against a variety of pathogens including bacteria, animals, and viruses (Suzuki, 2007).
Some of the lymphatic tissue associated with mucosa (MALT or GALT when referring specifically to the gut), forms prior to birth (e.g. Peyers patches) while other tissue can be induced in response to interactions with microbes (such as solitary follicles). Specific T cells and dendritic cells guide the switching of B cells to IgA production and B cells which produce IgA are attracted to mucous membranes while those which produce other antibody classes are not. Given the multiple independent pathways which result in IgA production in the gut, it is thought that this represents an ancestral feature of adaptive immunity (Suzuki, 2007).
1) LIGHT CHAINS
There are two supergenes/sets of genes which encode the mammalian light chains. Light chains are required for the expression of the heavy chains (Pitstrom, 2002). Mammals have two light chains, κ and λ which are thought to be derived from an ancestral λ chain. Shark light chains are similar to both κ and λ, although these two are very similar to each other. This suggests that the duplication of the light chains occurred before the evolution of the cartilaginous fish (Greenberg, 1993). The kappa sequences are on chromosome 2p12 and the lambda sequences are on chromosome 22q11. Both sets include constant, variable, and joining regions (neither possess the diversity regions present in the heavy chains) (OMIM). Three types of light chains are known in cartilaginous fish, bony fish, and amphibians. Birds are unusual in that they can mount good immune responses with only one VL and one VH family. (Lundqvist, 1999; Ishikawa, 2004). Antibodies which lack light chains exist in camels and some sharks (Conrath, 2003; Pitstrom, 2002).
--There are fewer than 50 VK (kappa variable) sequences (perhaps 15-20 is a more accurate estimate) that fall into one of 4 linked regions VK I through IV. VK pseudogenes exist on chromosomes 12, 15, and 22.
--There are more than 50 sequences in 800 kb of DNA. The variable sequences are divided into 3 clusters which contain the members of 9 families of sequences. The most frequently used sequences (cluster A, families 2 and 3) are those closest to the sequences for the joining and constant regions. There are at least 7 constant sequences of which at least 3 are pseudogenes.
Immmunoglobulin Iota chain
This immunoglobulin is conserved in mammals and is homologous to the lambda light chain. It is found associated with IgM on pre-B cells but not on mature B cells.
Immunoglobulin Omega Chain
The omega chain associates with IgM on pre-B cells but not on mature B cells. Mutations cause agammaglobulinemia.
B1 B cells can produce antibodies after interaction with macrophages or even with bacterial proteins. T-cells are not required for their antibody response and this mechanism may reflect the ancestral condition for the first adaptive immunity mechanisms (Martin, 2001). One group of B cells, B-1a B cells, must mature in the spleen before they are immunocompetent. They produce IgM, perhaps without exposure to the antigen, which binds to polysaccharides on the surface of bacteria and activates innate immunity mechanisms. The lack of these cells may be a factor in the susceptibility to infection by children born without a spleen (Wardemann, 2002).
Enhancers of antibody genes not only control
the expression of the mRNA, but also the recombination and class switching
(Magor, 1999). Amphibians were the first for class switching in which deletional recombination occurs at specific regions upstream
of the heavy chain genes (Stavnezer, 2004).
T cells are lymphocytes which mature in the thymus, as is evident in the following image of the thymus.
Most of the research on T cells has focused on the αβ T cells (which include the T helper and killer T cells) which bind to antigen presenting cells with antigens attached to their MHC proteins. Genetic analyses indicate that a second group of T cells which has not received as much attention, the γδ cells, is actually older than the αβ T cells and thus probably closer to the ancestral T cell state. While these γδ cells only compose 1-5% of the lymphocytes in blood, they can compose almost half of those found in other tissues, such as the gastrointestinal tract (such as in the Peyers patch in the following image). There are two types of T cell responses: Th1 seems to be a mammalian defense against intracellular parasites while Th2 seems to be a defense against nematode infections (Eriksson, 2004).
Marsupials produce a TCR chain (mu) which may be present in other groups of vertebrates as well. One copy of the gene apparently arose through retrotransposition and its regions are prejoined, unlike the typical T cell receptors (and the other copy of TCR mu in marsupials) which undergo somatic recombination to create diversity. It seems to be homologous to the delta chain produced in sharks (Parra, 2007).
The receptors of these γδ cells bind to antigens in a manner similar to the antibody receptors on B cells and do not require the processing of antigens by antigen presenting cells as do the αβ T cells (Richards, 2000).
T cells detect foreign and self peptides using a CD3/TcR complex. The CD3 complex is composed of five chains (γ, ε, ζ, η, and δ) which are not polymorphic (Fellah, 2002). Humans have four TCR chains, alpha, beta, gama, and delta, which combine in the thymus to form alpha-beta or gamma-delta heterodimers (Parra, 2007).
The immunoglobulin light chain and the T cell receptors seem to have been derived from the same precursor molecule early in vertebrate history, perhaps in one of the polyploidy events early in vertebrate evolution. Light antibody chains became associated with heavy antibody chains and the TcRα became associated with the β chain (Pitstrom, 2002).
CD8 and CD4 are coreceptors which function with TcR and are not expressed on the same classes of T cells. CD8 interacts with MHC I molecules and CD4 interacts with MHC II molecules (Fellah, 2002).
While most CD8 cells
express αβ TcRs,
some express αα
TcR receptors (such as NK cells, some dendritic
cells, some γδ T cells, and intra-epithelial
lymphocytes). CDα subunits are known in fish and CDβ
subunits are known in amphibians (Fellah,
TCR receptors a subunits are composed of V, J, and C regions while b subunits are composed of V, D, J, and C regions. The heterodimer composed of a and b subunits form the receptors on T helper and cytotoxic T cells. The recombination of TCR sequences produces an estimated 1015 different types of T cell receptors.
The sequences of TCRa are located on chromosome 14q11. There are more than 100 sequences known and many more may exist.
The sequences of TCRb are located on chromosome 7q22. There are more than 59 sequences in the cluster.
TCRd is composed of V, D, J, and C regions. The variable region can contain up to 4 variable segments, more than in any other known antibody or T cell receptor. Interestingly, the delta sequences are located in the middle of the TCRa sequences (in the order Va--Vd--Dd--Jd--Cd--Ja--Ca).
The 20 or so TCRg sequences are located in a 160 kb stretch of DNA on chromosome 7p15. There are V, J, and C sequences and pseudogenes in the following order: