**6. The value of gene duplication to adaptive immunity**

### **6.1 Adaptive versus innate immunity**

The innate immune system is the "first responder" element of immune protection for higher vertebrates and may be the sole system for most invertebrate. Innate immunity works through phagocytic and epithelial cells that bear so-called "innate immune receptors", e.g. Toll-like (TLR) that recognize bacterial and viral entities that are not produced by eucaryotic cells of the host and are therefore considered foreign and dangerous. For vertebrates these include lipopolysaccharide, flagellin, bacterial DNA (non-methylated) and double-strained RNA. These are referred to as PAMPs (**P**athogen **A**ssociated **M**olecular **P**atterns). Products liberated from cells stimulated when their innate receptors recognize these PAMPs, then stimulate lymphocytes that leads to development of the adaptive immune system. This is demonstrated in studies using the isolator piglet model in which colonization or purified PAMPs are required for an adaptive immune response (Butler et al., 2002; 2005) and by infection with influenza virus which generates double-stranded RNA during replication (Butler, Lager, Vincent, Sun unpublished).

Unlike antibodies, the ligand binding sites of innate immune receptors do not change their specificity by any somatic process when they encounter a PAMP. The capacity for receptor modification after antigen encounter is the property of the adaptive immune system, as implied by the name. This adaptive capacity is illustrated in Figure 9 by showing there is a profound increase in Ig secretion and a shift in isotype usage (Fig. 9A) and an increase in SHM of the adaptive immune system antigen receptors after antigen exposure (Fig. 9B). As illustrated, these somatic hypermutations accumulate in those regions of the VH genes that encode the antibody binding site, i.e. the CDRs (Fig. 9C; Fig. 1A).

### **6.2 Are multiple VH genes required for host immune protection?**

Since swine use a very small number of VH genes to generate a VDJ repertoire capable of protecting the host at all ages (Sun et al., 1998; Butler et al., 2006), we did a statistical itineration to suggest that >95% of the adaptive VDJ repertoire was the result of diversity within HCDR3 (Butler et al., 2000). HCDR3 is not encoded by any particular V-region gene segment but rather by the recombination of VH-DH-JH (Fig. 7 & 8). Joining of VH-DH-JH involves exonuclease removal of nucleotides from the gene segments involved as well as nucleotide additions using deoxynucleotide transferase. The types of variations generated are illustrated in Fig. 8 which shows different HCDR3 sequences found among the recombinants that use just one VH, DH and JH segment. The importance of the diversity generated in HCDR3 was empirically shown by Xu and Davis (2000) to be sufficient to allow most adaptive immune response using a transgenic mouse given only on VH gene but an intact DH and JH genome. Studies in rabbit also show that a single VH gene is primarily used in the establishment of the antibody repertoire (Knight 1992) but following antigen encounter this VH gene can be modified by SHM as well as by SGC

Immunoglobulin Polygeny: An Evolutionary Perspective 129

There are various examples of polygeny in the immune system but in these loci SHM is not important in repertoire formation. The loci encoding the various T cell receptors are similar to those encoding the Igs. Recombination of gene segments occurs in the same manner as for Ig genes, i.e. recombination requires RAGs, DNA excision, and splicing and DNA repair enzymes(see Fig. 7). However there is no convincing evidence for SHM after segment recombination so polygeny in the TCR is theoretically more important in repertoire

The genes encoding the major histocompatibility genes (MHC) are partially encoded by IGSF genes and determine: (a) "individualism" and self recognition as demonstrated in tissue typing and (b) recognition of peptides generated and presented by antigen-presenting cells. The MHC gene system is highly conserved among mammals with three genes encoding MHC I and 6-8 encoding MHC II. The enormous diversity of MHC is not achieved by polygeny or somatic processes but rather by an enormous degree of polymorphism of the MHC genes within the population (Janeway et al., 2005). There can be as many as 300 allelic

The selective advantage of polygeny for genes encoding the MHC, innate immune receptors, the CH and C subclasses and even the TCR is easy to appreciate. However, such polygeny in loci encoding the variable Ig genes in higher vertebrates is more difficult to explain if these species can generate an effective antibody response using a single VH gene (Section 6.2). This creates an enigma for VH and VL polygeny that we shall attempt to explain from

**7. Conclusions: Ig polygeny and redundancy in higher vertebrates is an** 

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.,

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).

**6.4 Polygeny is widespread in other loci important to immunity** 

variants of any one MHC gene.

an evolutionary perspective.

**7.1 The case of mammalian IgD** 

1983; Liu et al., 1996; Roes et al., 1993; Vitetta et al., 1977).

**evolutionary vestige** 

generation than in the Ig loci where SHM can further diversify the repertoire.

(Winstead et al., 1999; Schiaffella et al., 1999). We show that in swine seven VH genes can account for 93% of the entire pre-repertoire and that the two duplicated VHA genes (which have idetntical CDR1 and CDR regions; Fig. 4) can alone account for 30-50% (Fig. 10). Collectively these studies raise the question as to why the VH repertoire has been so heavily duplicated while so few of these duplicons are used. The answer may be found among bats, or at least in the insectivorous microbats. *Myotis lucifugus* has >250 VH3 family genes (Fig. 5) and probably 350 total VH genes, including all families (Bratsch et al., 2011). However, SHM occurs at the frequency seen in fetal piglets (Fig. 9B). Perhaps some older mammalian orders like the Chiroptera, rely more heavily on VH polygeny than somatic modification.

### **6.3 How does duplication/ diversification in the C-region effect protective immunity**

The survival value of C-region polygeny can be appreciated because antibody isotypes encoded by the exons for IgM, IgG etc have distinct biological functions (Janeway et al., 2005). Additional duplication of C and C genes has generated modified duplicons encoding Ig subclasses also with different important biological functions. In the case of IgG subclasses, these involve features like the ability to be recognized by different Fc receptors on phagocytic cells, transport across epithelial barriers, serum half-life differences involving FcRn and an association with antibodies of certain specificities. Duplication of C genes in cattle (and other domestic ruminants) has lead to subclass IgG1 that is selectively transported by acinar epithelial cells of the mammary gland to provide essential antibodies for the survival of the newborn (Butler 1974; 1998). Neither IgG2 nor IgG3 function in this capacity. While there have been no functional studies on the many IgG subclass antibodies in horse or swine, it is possible that each of these subclass antibodies are best suited for particular activities in the same manner as described for human IgG subclasses.

The duplicated human C genes also differ in a number of features. Most striking is the susceptibility of IgA1 to IgA proteases produced by many common Gram positive bacteria while IgA2 is resistance due to the lack of the 13 amino acid hinge which is the substrate for these proteases in IgA1 (Plaut et al., 1974). Differential tissue expression of rabbit IgA subclasses also suggests a division of labor ( Spieker-Polet et al., 1993). IgA in swine lack the long hinge of human IgA1 and is therefore not susceptible to the classical bacterial IgA1 proteases although a protease from *H. suis* can cleave the porcine -chain (M. Mullins, K. Register, D.O. Bayles, J.E. Butler, unpub).

The "experiment of nature" is whether mammals with a deficiency in their C sublocus are immunologically impaired. There are no known ruminates that like lack ruminat IgG1, so there are no data; perhaps such a deficiency would be a developmental lethal. The mammals best-studied for C subclass deficiencies are humans. For example, humans lacking IgG2 have a deficiency in their response to bacterial polysaccharide antigens (Hammarstrom et al., 1986). Additional deletions of C genes have been described, including individuals lacking C1, C2, C4 and C1 (the 5'duplicon shown in Figure 2B). However, such individuals remain healthy and asymptomatic (Lefranc et al., 1983b; Notoaramgelo et al., 2009). Selective IgG1 deficiciency which accounts for the major portion of serum IgG, is not correlated with lower serum igG levels (Olsson et al., 1993) while some appear to be (Rabbani et al., 1995). In swine, the IgAb allotypic variant lacks a major portion of its hinge (Brown et al., 1995), yet this "defect" has not been correlated with any risk of disease; (Navarro et al., 2000).

(Winstead et al., 1999; Schiaffella et al., 1999). We show that in swine seven VH genes can account for 93% of the entire pre-repertoire and that the two duplicated VHA genes (which have idetntical CDR1 and CDR regions; Fig. 4) can alone account for 30-50% (Fig. 10). Collectively these studies raise the question as to why the VH repertoire has been so heavily duplicated while so few of these duplicons are used. The answer may be found among bats, or at least in the insectivorous microbats. *Myotis lucifugus* has >250 VH3 family genes (Fig. 5) and probably 350 total VH genes, including all families (Bratsch et al., 2011). However, SHM occurs at the frequency seen in fetal piglets (Fig. 9B). Perhaps some older mammalian orders like the Chiroptera, rely more heavily on VH polygeny

**6.3 How does duplication/ diversification in the C-region effect protective immunity**  The survival value of C-region polygeny can be appreciated because antibody isotypes encoded by the exons for IgM, IgG etc have distinct biological functions (Janeway et al., 2005). Additional duplication of C and C genes has generated modified duplicons encoding Ig subclasses also with different important biological functions. In the case of IgG subclasses, these involve features like the ability to be recognized by different Fc receptors on phagocytic cells, transport across epithelial barriers, serum half-life differences involving FcRn and an association with antibodies of certain specificities. Duplication of C genes in cattle (and other domestic ruminants) has lead to subclass IgG1 that is selectively transported by acinar epithelial cells of the mammary gland to provide essential antibodies for the survival of the newborn (Butler 1974; 1998). Neither IgG2 nor IgG3 function in this capacity. While there have been no functional studies on the many IgG subclass antibodies in horse or swine, it is possible that each of these subclass antibodies are best suited for

particular activities in the same manner as described for human IgG subclasses.

The duplicated human C genes also differ in a number of features. Most striking is the susceptibility of IgA1 to IgA proteases produced by many common Gram positive bacteria while IgA2 is resistance due to the lack of the 13 amino acid hinge which is the substrate for these proteases in IgA1 (Plaut et al., 1974). Differential tissue expression of rabbit IgA subclasses also suggests a division of labor ( Spieker-Polet et al., 1993). IgA in swine lack the long hinge of human IgA1 and is therefore not susceptible to the classical bacterial IgA1 proteases although a protease from *H. suis* can cleave the porcine -chain (M. Mullins, K.

The "experiment of nature" is whether mammals with a deficiency in their C sublocus are immunologically impaired. There are no known ruminates that like lack ruminat IgG1, so there are no data; perhaps such a deficiency would be a developmental lethal. The mammals best-studied for C subclass deficiencies are humans. For example, humans lacking IgG2 have a deficiency in their response to bacterial polysaccharide antigens (Hammarstrom et al., 1986). Additional deletions of C genes have been described, including individuals lacking C1, C2, C4 and C1 (the 5'duplicon shown in Figure 2B). However, such individuals remain healthy and asymptomatic (Lefranc et al., 1983b; Notoaramgelo et al., 2009). Selective IgG1 deficiciency which accounts for the major portion of serum IgG, is not correlated with lower serum igG levels (Olsson et al., 1993) while some appear to be (Rabbani et al., 1995). In swine, the IgAb allotypic variant lacks a major portion of its hinge (Brown et al., 1995), yet this "defect" has not been correlated with any risk of

than somatic modification.

Register, D.O. Bayles, J.E. Butler, unpub).

disease; (Navarro et al., 2000).

## **6.4 Polygeny is widespread in other loci important to immunity**

There are various examples of polygeny in the immune system but in these loci SHM is not important in repertoire formation. The loci encoding the various T cell receptors are similar to those encoding the Igs. Recombination of gene segments occurs in the same manner as for Ig genes, i.e. recombination requires RAGs, DNA excision, and splicing and DNA repair enzymes(see Fig. 7). However there is no convincing evidence for SHM after segment recombination so polygeny in the TCR is theoretically more important in repertoire generation than in the Ig loci where SHM can further diversify the repertoire.

The genes encoding the major histocompatibility genes (MHC) are partially encoded by IGSF genes and determine: (a) "individualism" and self recognition as demonstrated in tissue typing and (b) recognition of peptides generated and presented by antigen-presenting cells. The MHC gene system is highly conserved among mammals with three genes encoding MHC I and 6-8 encoding MHC II. The enormous diversity of MHC is not achieved by polygeny or somatic processes but rather by an enormous degree of polymorphism of the MHC genes within the population (Janeway et al., 2005). There can be as many as 300 allelic variants of any one MHC gene.

The selective advantage of polygeny for genes encoding the MHC, innate immune receptors, the CH and C subclasses and even the TCR is easy to appreciate. However, such polygeny in loci encoding the variable Ig genes in higher vertebrates is more difficult to explain if these species can generate an effective antibody response using a single VH gene (Section 6.2). This creates an enigma for VH and VL polygeny that we shall attempt to explain from an evolutionary perspective.
