**5. Patterns of V, D and J gene segment usage**

### **5.1 VH usage is biased to favor certain VH genes**

Table 1 shows that higher vertebrates have many duplicated V-region gene segments available for use in the formation of their antibody repertoire using the recombinatorial process illustrated in Figure 7. Humans have available ~ 100 VH segments, ~30 DH segments and 9 JH segments (Fig. 2A). By contrast, swine have <30 VH genes belonging to a single family (Fig. 4), only two functional DH segments and like the chicken (Fig. 3) one functional JH segment (Sun et al., 1994; Butler et al., 1996; Eguchi-Ogawa et al., 2010). While the ancestral VH3 family (Schroeder et al., 1990) dominates the V-region loci of many species, the ~100 VH genes of mice and human belong to 14 and 7 different families respectively (Table 1).

Usage of VH genes in rabbit is biased to the most 3' VH gene, which accounts for 90% of VH usage in the pre-immune repertoire although there are >100 VH genes in the rabbit repertoire (Currier et al., 1988; Table 2). In humans there is bias for V3-23,V3-30,V3-33 and V4-34 (Glas et al., 2000). While some suggest that VH usage is random in mice (Dildrop et al., 1985) studies on J558 usage (one-half of the mouse genome) indicates that usage in unequal and rather scattered across the entire J558 genome even in the pre-immune repertoire (Gu et al., 1991) and that usage is not affected by SHM or CSR. Foster et al., (1997) showed that while most Vk genes were used, usage was non-random and the same was true for J. Sheehan et al., (1993) showed that fetal VH usage can differ from 0.1 to 1.0 but that most 5' VH genes are underrepresented. In swine VHA (IGHV4) and its near duplicate (IGHV10; see Figs. 4 & 10) account for one-third to one-half of the pre-immune repertoire (Butler et al., 2006; Eguchi-Ogawa et al., 2010; Butler et al., 2011b). Interestingly, the majority of these preferred genes in all these species belong to the ancestral VH3 family (Schroder et al., 1990; Brezinschek et al., 1997).

### **5.2 Variable region gene segment usage is not position dependent**

Early studies suggested that VH usage was biased during early stages of B cell lymphogenesis to favor the most JH proximal DH segments and the most 3' VH genes (Schroeder et al., 1987; Yancopoulos et al., 1984) but that this pattern became "normalized" in adults (Malynn et al., 1987). This concept gained support when it was found that young rabbits use their 3' most VH gene > 90% of the time and then further diversfied their repertoire using upstream VH genes and SGC; perhaps a type of "developmental normalization (Knight 1992; Becker & Knight 1990). However, additional studies in humans neither substantiated the positional "3' bias" (Matsuda et al., 1993) nor have our studies in swine (Eguchi-Ogawa et al., 2010; Fig. 10). The most 3' functional VH in swine (IGHV2) is almost never used while upstream VH15 (IGHV15) can account for ~13% of VH usage (Fig. 10). Thus, the "position hypothesis" to explain VH usage has not been universally fulfilled.

### **5.3 VH gene usage remains constant in fetal and young pigs**

Vertical studies on VH usage in especially humans and mice are difficult because: (a) the V-D-J repertoire of these species is complex (Table 2) and could require up to 56 primer sets to recover all VDJ rearrangements in mouse and 42 sets for human (b) maternal regulatory factors transmitted *in utero* or via colostrum/milk can influence pre-natal and postnatal development (Wikler et al., 1980; Rodkey & Adler, 1983; Klobasa et al., 1981; Wang & Shlomchik 1998; Yamaguchi et al., 1983) and (c) control of environmental and maternal

Immunoglobulin Polygeny: An Evolutionary Perspective 127

discussed in the last section, the bias usage of certain VH genes reported for these species (Section 5.1) suggests the outcome might be similar if such studies could be efficiently performed. The answer to the question may reside in understanding the evolution of the genes encoding antibody specificity that generated the vast array of Ig variable region genes first seen in more primitive vertebrates and the later evolution of somatic events that would

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

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

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

appear to have made the original polygeny unnecessary.

**6.1 Adaptive versus innate immunity** 

(Butler, Lager, Vincent, Sun unpublished).

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

encode the antibody binding site, i.e. the CDRs (Fig. 9C; Fig. 1A).

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

effects is difficult or impossible in species with altricial offspring. Therefore we addressed this issue using a piglet model in which there is no transfer of maternal factors *in utero* and the influence of environmental factors postnatally on their precosial offspring can be controlled by the experimenter (Butler & Sinkora 2007; Butler et al., 2009b). Use of this model revealed that VH usage was constant during fetal life and that seven major genes accounted for >90% of the repertoire (Fig. 10; Butler et al. 2011b) while four can explain >80% of the repertoire Interestingly in piglets exposed to viral infection, gut colonization or nematode parasites after birth, ~ 75% of the mutated genes used were the same seven (Fig. 10; extreme right). Furthermore, proportional usage of these genes was similar to what was seen in the pre-immune repertoire, albeit somatically mutated. Some modest changes were observed such as an increase in VHY and decreases in VHA\* and VHN. In other words, swine seldom select other genes from their repertoire after exposure to environmental antigen, but continue to use the same VH genes that comprise ~93% of the pre-immune repertoire.

Fig. 10. VH gene usage in fetal piglets and among neonates that are antigen-exposed remains relatively constant. DG= days of gestation. The number of VH gene clones tested is given in the legend. The mutated VH genes are no longer recognized because they do not hybridize with VH gene-specific probes. Their identity must then be determined by sequencing. The bar graph on the extreme right gives the proportion of all mutated VH genes that are accounted for by the major seven genes used in the pre-immune repertoire by the fetus.

These observations should not be surprising considering that the specificity of binding site is heavily dependent on HCDR3 (Section 6.2). The HCDR3 repertoire in swine is diverse when only one VH, one DH and one JH segment are used (Fig. 8). Furthermore, the limited use of VH genes should not limit specificity, since their CDR regions can also be somatically mutated (Fig. 9B, C). As discussed in 4.1, the variants from a single VH (Fig. 8) can pair with different light chains, which can further reduce the need for large numbers of different VH genes for repertoire formation.

### **5.4 Why is the germline VH repertoire large but usage of the repertoire limited?**

Use of the piglet model demonstrates that most of the piglet VH repertoire (Fig. 4 versus Fig. 10) is seldom used to form the antibody repertoire. While corresponding vertical studied are lacking in mice and human because of the logistic and experimental reasons

effects is difficult or impossible in species with altricial offspring. Therefore we addressed this issue using a piglet model in which there is no transfer of maternal factors *in utero* and the influence of environmental factors postnatally on their precosial offspring can be controlled by the experimenter (Butler & Sinkora 2007; Butler et al., 2009b). Use of this model revealed that VH usage was constant during fetal life and that seven major genes accounted for >90% of the repertoire (Fig. 10; Butler et al. 2011b) while four can explain >80% of the repertoire Interestingly in piglets exposed to viral infection, gut colonization or nematode parasites after birth, ~ 75% of the mutated genes used were the same seven (Fig. 10; extreme right). Furthermore, proportional usage of these genes was similar to what was seen in the pre-immune repertoire, albeit somatically mutated. Some modest changes were observed such as an increase in VHY and decreases in VHA\* and VHN. In other words, swine seldom select other genes from their repertoire after exposure to environmental antigen, but continue to use the same VH genes that comprise ~93% of the pre-immune

**VHG VHA VHB VHE VHA\* VHF VHY VHN/VHZZ VHC VHZ % Major** 

 **(VH2) (VH4) (VH6/ VH12) (VH8) (VH10) (VH11) (VH14) (VH15) (VH?) (VH?) (seven)**

These observations should not be surprising considering that the specificity of binding site is heavily dependent on HCDR3 (Section 6.2). The HCDR3 repertoire in swine is diverse when only one VH, one DH and one JH segment are used (Fig. 8). Furthermore, the limited use of VH genes should not limit specificity, since their CDR regions can also be somatically mutated (Fig. 9B, C). As discussed in 4.1, the variants from a single VH (Fig. 8) can pair with different light chains, which can further reduce the need for large numbers of different

**5.4 Why is the germline VH repertoire large but usage of the repertoire limited?**  Use of the piglet model demonstrates that most of the piglet VH repertoire (Fig. 4 versus Fig. 10) is seldom used to form the antibody repertoire. While corresponding vertical studied are lacking in mice and human because of the logistic and experimental reasons

**Mutated in antigen-exposed piglets(136)**

**20-50DG (2108) 95DG (3357)**

Fig. 10. VH gene usage in fetal piglets and among neonates that are antigen-exposed remains relatively constant. DG= days of gestation. The number of VH gene clones tested is given in the legend. The mutated VH genes are no longer recognized because they do not hybridize with VH gene-specific probes. Their identity must then be determined by sequencing. The bar graph on the extreme right gives the proportion of all mutated VH genes that are accounted for by the major seven genes used in the pre-immune repertoire by

repertoire.

the fetus.

VH genes for repertoire formation.

**Proportional of Usage**

discussed in the last section, the bias usage of certain VH genes reported for these species (Section 5.1) suggests the outcome might be similar if such studies could be efficiently performed. The answer to the question may reside in understanding the evolution of the genes encoding antibody specificity that generated the vast array of Ig variable region genes first seen in more primitive vertebrates and the later evolution of somatic events that would appear to have made the original polygeny unnecessary.
