**7.1 The case of mammalian IgD**

Vestiges of genes are not unusual and mammalian IgD is an example. IgD was discovered as a myeloma protein nearly 50 years ago and its function has remained an enigma since that time (Rowe & Fahey, 1965). The considerable research funding invested to determine the function of IgD has largely generated only hypotheses. IgD is the least homologous isotype among mammals, e.g. <40%,(in part the reason it was overlooked in some mammals; Butler et al., 1996) whereas most other isotypes share 70-90% homology (Butler, 2006). IgD is even missing from the genome of some mammals (Table 2; Fig. 2B) and perhaps all birds. In mice and humans, IgD and IgM occur as dual B cell receptors but IgD-deficient mice have normal immune responses (Nitschke et al., 1993) although IgD can compensate for the loss of functional IgM (Lutz et al., 1998). While numerous studies have attempted to define a unique role for mammalian IgD, most of these have not been very convincing (Monroe et al., 1983; Liu et al., 1996; Roes et al., 1993; Vitetta et al., 1977).

Comparative immunologists have put the role of IgD into perspective beginning with the observation in catfish of a seven domain Ig with distant homology to mammalian IgD (Wilson et al., 1997; Bengten et al., 2002). This was followed by the discovery of a similar multi-domain IgD in *Xenopus* (Zhao et al., 2006) and in other teleosts (Hordvik et al., 1999; Srisapoome et al., 2004; Stenvik & Jorgensen, 2000). IgD has subsequently been found in the genome of many other lower vertebrates and in protherian mammals (reviewed by Edholm et al., 2010). Collectively these studies would morph into the realization that IgD and IgM are the primordial vertebrates Ig isotypes (Ohta & Flajnik, 2006; Bengten et al., 2006).

Immunoglobulin Polygeny: An Evolutionary Perspective 131

fact that >90% of the repertoire is generated by junctional diversity in HCDR3 (Section 6.2; Fig. 8) and that a transgenic mouse with only one VH is fully immune competent, strengthens the argument. Studies in rabbit also support this view in which one VH gene account for 90% of the early repertoire which can be diversified SHM and SGC after antigen encounter (Winstead et al., 1999; Schiaffella et al., 1999). Detractors from this view may argue that while few (or only one) VH gene are needed, polygeny in the DH and JH regions is still necessary (Table 1). Again studies in swine counter this argument since they have only two functional DH segments and one JH (Butler et al., 1996; Eguchi-Ogawa et al., 2010). In addition, the chicken has only a single JH, DH segments that are nearly identical and only one functional VH and one function V gene (Fig.3). This species uses the "relics" of upstream pseudogenes for use in repertoire diversification by SGC (Reynaud et al., 1987; Ratcliffe 2006), a mechanism also available to rabbits (Becker & Knight, 1990). Thus we propose that the evolution of gene segment recombination and SHM rendered polygeny in the VH and VL loci of higher vertebrates unnecessary. We believe this polygeny is derived from the tandem array of V-D-J-C clusters in sharks that do not require somatic recombination (Fig. 3). Sharks lack germinal centers as do bony fished and amphibians which makes SHM a less efficient process (DuPasquir et al., 2000). While SHM had been described in these vertebrate classes, we cannot cite head-to-head studies on the frequency

We believes that like IgD, the VH, V and V polygeny of the most evolved mammals and birds, remain in the genome largely as relics and because of the lack of negative selection. Early vertebrates have as many as four light chain loci but with evolution the number diminished and birds have lost all but lambda. That different Ig isotypes and certain IgG subclass have specific biological functions suggests a selective advantage for polygeny which we believe was true at the dawn of VH and VL duplicatation that lead to the polygeny in V-region loci. The alternative explanation for especially VH and VL polygeny is that this polygeny occurs in a "hot spot" of RAG-dependent gene segment recombination. For such recombination events to occur, opening of the chromatin structure to provide access to nuclear enzymes, is considered necessary. Perhaps such exposure to repeated recombination activity explains the instability of the locus (Lefranc et al 1983a; Matsuda et al 1990) which renders genes in the locus vulnerable to the molecular machinery involved in

Mainstream immunology has invested almost entirely in studies of mouse and human immune systems and therefore seems to have missed the evolutionary significance of Ig polygeny. To avoid a similar criticism our analysis of polygeny reviewed the major elements of the Ig genes of all vertebrates that have been seriously studied and the mechanisms they use to generate their antibody repertoire. From these comparisons we have offered a hypothesis to explain the polygeny of the major Ig loci in mammals and the reason why at the "highest levels" of vertebrate development, this polygeny appears to be an evolutionary

Gene duplication is a common feature of eucaryotic genomes although the degree varies among gene families and loci. It is estimated that ~5% of the human genome is comprised of duplicated genes (Lewin, 2004). Among these are genes of the immunoglobulin supergene family (IGSF). This polygeny is widespread in loci important to the immune system as well as

of SHM like that presented in Fig. 9B.

gene duplication and genomic gene conversion.

vestige.

**8. Conclusions** 

Interesting among the studies in catfish and swine, is that IgD can be produced as a chimeric Ig using the C1 domain of IgM and the various domain exons of C (Zhao et al., 2002; 2003). Thus, the expression of IgD in mice and humans by RNA splicing rather than classical CSR (Maki et al., 1981) has a primitive history.

Compared to the IgD of fish and *Xenopus*, the exons encoding for mammalian IgD appear to be relics. Depending on the mammal selected, the number of hinge and domain exons is highly variable (Butler et al., 2010). While switch regions are typically >3kb in length, mammalian S (when present) exists as a short remnant of <0.5 kb (Zhao et al., 2003). Nevertheless it appears to function in some cases in CSR in humans and swine (Zhao et al., 2002; 2003; Arpin et al., 1998; Koelsch et al., 2007; Zheng et al., 2004).

Recent findings show that basophils have abundant membrane IgD but not T cells, NK cells, dendritic cells or monocytes (Chen et al., 2009; Dawichi & Marshall, 2007) although this observation was not supported by the recent report by Karasuyami et al., (2009). Assuming the case for adventious IgD on basophils is true, it agrees with studies in catfish showing surface IgD on granulocytes (Edholm et al., 2010). These observations would support a unique role of this ancestral Ig. In spite of the observation of IgD on human basophils, the "experiment of nature" that IgD deficiency does not impair mammalian adaptive immunity (Nitschke et al., 1993) has lead us to conclude that IgD is an evolutionary relic for mammals but persists because of its redundant value including its role as a BCR and its presence as an adventious Ig on basophils (Lutz et al., 1998; Chen et al., 2009). Thus, the relic remains because there has been no negative selection to completely remove IgD from most mammalian genomes.

### **7.2 CH duplication/diversification provides antibodies with specialized effector function but also redundancy**

Isotype diversity in sharks and bony fish is limited to IgM and IgD (IgW in sharks). In tetrapods the CH repertoire diversified; 3 in birds and reptiles, 5-6 in amphibians and 5 in mammals (Table 2). In mammals this includes subclasses of IgG and IgA. Each major isotypes in mammals, perhaps with the exception of IgD, has some specialized effector function (Janeway et al., 2005). Perhaps the terrestrial environment offered a new challenge to survival and with the addition of homeothermia, the need for a more specialized adaptive immune system that could respond more quickly and lead to the evolution of GCs (Sections 3.2; 6.3). As discussed in Section 3-2, there is also evidence that the polygeny of Cgenes resulted from a combination of gene duplication and genomic gene conversion. This duplication event was restricted to mammals that appeared in the "last minute before 12PM" on the evolutionary clock, appearing after mammalian speciation (Butler et al., 2009a). The subclass duplication/diversification in mammals resembles the pattern that produced V region polygeny. In sharks and bony fishes, isotype diversity is limited to IgM and IgD, while in higher vertebrates duplication/ diversification extends downstream into the CH sublocus, and in mammals, especially to the C sublocus. However, IgG subclass deficiencies are not lethal defects, suggesting that even in late-evolving sites of Ig gene duplication, such duplication seem unnecessary.

### **7.3 Somatic mechanisms render much of the polygeny in VH, V and V loci to relics**

As we show in the piglet model, very few VH genes are needed and after antigen encounter, further repertoire diversification is by SHM of the same genes (Fig. 10). Furthermore, the

Interesting among the studies in catfish and swine, is that IgD can be produced as a chimeric Ig using the C1 domain of IgM and the various domain exons of C (Zhao et al., 2002; 2003). Thus, the expression of IgD in mice and humans by RNA splicing rather than classical

Compared to the IgD of fish and *Xenopus*, the exons encoding for mammalian IgD appear to be relics. Depending on the mammal selected, the number of hinge and domain exons is highly variable (Butler et al., 2010). While switch regions are typically >3kb in length, mammalian S (when present) exists as a short remnant of <0.5 kb (Zhao et al., 2003). Nevertheless it appears to function in some cases in CSR in humans and swine (Zhao et al.,

Recent findings show that basophils have abundant membrane IgD but not T cells, NK cells, dendritic cells or monocytes (Chen et al., 2009; Dawichi & Marshall, 2007) although this observation was not supported by the recent report by Karasuyami et al., (2009). Assuming the case for adventious IgD on basophils is true, it agrees with studies in catfish showing surface IgD on granulocytes (Edholm et al., 2010). These observations would support a unique role of this ancestral Ig. In spite of the observation of IgD on human basophils, the "experiment of nature" that IgD deficiency does not impair mammalian adaptive immunity (Nitschke et al., 1993) has lead us to conclude that IgD is an evolutionary relic for mammals but persists because of its redundant value including its role as a BCR and its presence as an adventious Ig on basophils (Lutz et al., 1998; Chen et al., 2009). Thus, the relic remains because there has been no negative selection to completely remove IgD from most

**7.2 CH duplication/diversification provides antibodies with specialized effector** 

Isotype diversity in sharks and bony fish is limited to IgM and IgD (IgW in sharks). In tetrapods the CH repertoire diversified; 3 in birds and reptiles, 5-6 in amphibians and 5 in mammals (Table 2). In mammals this includes subclasses of IgG and IgA. Each major isotypes in mammals, perhaps with the exception of IgD, has some specialized effector function (Janeway et al., 2005). Perhaps the terrestrial environment offered a new challenge to survival and with the addition of homeothermia, the need for a more specialized adaptive immune system that could respond more quickly and lead to the evolution of GCs (Sections 3.2; 6.3). As discussed in Section 3-2, there is also evidence that the polygeny of Cgenes resulted from a combination of gene duplication and genomic gene conversion. This duplication event was restricted to mammals that appeared in the "last minute before 12PM" on the evolutionary clock, appearing after mammalian speciation (Butler et al., 2009a). The subclass duplication/diversification in mammals resembles the pattern that produced V region polygeny. In sharks and bony fishes, isotype diversity is limited to IgM and IgD, while in higher vertebrates duplication/ diversification extends downstream into the CH sublocus, and in mammals, especially to the C sublocus. However, IgG subclass deficiencies are not lethal defects, suggesting that even in late-evolving sites of Ig gene

**7.3 Somatic mechanisms render much of the polygeny in VH, V and V loci to relics**  As we show in the piglet model, very few VH genes are needed and after antigen encounter, further repertoire diversification is by SHM of the same genes (Fig. 10). Furthermore, the

CSR (Maki et al., 1981) has a primitive history.

mammalian genomes.

**function but also redundancy** 

duplication, such duplication seem unnecessary.

2002; 2003; Arpin et al., 1998; Koelsch et al., 2007; Zheng et al., 2004).

fact that >90% of the repertoire is generated by junctional diversity in HCDR3 (Section 6.2; Fig. 8) and that a transgenic mouse with only one VH is fully immune competent, strengthens the argument. Studies in rabbit also support this view in which one VH gene account for 90% of the early repertoire which can be diversified SHM and SGC after antigen encounter (Winstead et al., 1999; Schiaffella et al., 1999). Detractors from this view may argue that while few (or only one) VH gene are needed, polygeny in the DH and JH regions is still necessary (Table 1). Again studies in swine counter this argument since they have only two functional DH segments and one JH (Butler et al., 1996; Eguchi-Ogawa et al., 2010). In addition, the chicken has only a single JH, DH segments that are nearly identical and only one functional VH and one function V gene (Fig.3). This species uses the "relics" of upstream pseudogenes for use in repertoire diversification by SGC (Reynaud et al., 1987; Ratcliffe 2006), a mechanism also available to rabbits (Becker & Knight, 1990). Thus we propose that the evolution of gene segment recombination and SHM rendered polygeny in the VH and VL loci of higher vertebrates unnecessary. We believe this polygeny is derived from the tandem array of V-D-J-C clusters in sharks that do not require somatic recombination (Fig. 3). Sharks lack germinal centers as do bony fished and amphibians which makes SHM a less efficient process (DuPasquir et al., 2000). While SHM had been described in these vertebrate classes, we cannot cite head-to-head studies on the frequency of SHM like that presented in Fig. 9B.

We believes that like IgD, the VH, V and V polygeny of the most evolved mammals and birds, remain in the genome largely as relics and because of the lack of negative selection. Early vertebrates have as many as four light chain loci but with evolution the number diminished and birds have lost all but lambda. That different Ig isotypes and certain IgG subclass have specific biological functions suggests a selective advantage for polygeny which we believe was true at the dawn of VH and VL duplicatation that lead to the polygeny in V-region loci. The alternative explanation for especially VH and VL polygeny is that this polygeny occurs in a "hot spot" of RAG-dependent gene segment recombination. For such recombination events to occur, opening of the chromatin structure to provide access to nuclear enzymes, is considered necessary. Perhaps such exposure to repeated recombination activity explains the instability of the locus (Lefranc et al 1983a; Matsuda et al 1990) which renders genes in the locus vulnerable to the molecular machinery involved in gene duplication and genomic gene conversion.

Mainstream immunology has invested almost entirely in studies of mouse and human immune systems and therefore seems to have missed the evolutionary significance of Ig polygeny. To avoid a similar criticism our analysis of polygeny reviewed the major elements of the Ig genes of all vertebrates that have been seriously studied and the mechanisms they use to generate their antibody repertoire. From these comparisons we have offered a hypothesis to explain the polygeny of the major Ig loci in mammals and the reason why at the "highest levels" of vertebrate development, this polygeny appears to be an evolutionary vestige.
