ADAPTIVE IMMUNITY

3.1 T-cells and receptors

The TCR are essential in antigen recognition and acti­vation of the adaptive immune response. There are four conventional TCR chains conserved across all jawed vertebrates: α, β, γ and δ.

For decades it was accepted that only four TCR chains existed, until the discovery of a fifth (TCRμ) in the northern brown bandicoot (Isoodon macrourus) (Baker et al. 2005). TCRμ is an ancient TCR chain that has been maintained in monotremes and marsupials but lost in eutherian mammals during evolution (Wang et al. 2011). Structurally, the C domain of TCRμ is simi­lar to TCRδ, but the V domain more closely matches those of immunoglobulin heavy chains, suggesting that TCRμ arose from recombination between a TCR and an immunoglobulin (Miller 2010). TCRμ diversity within the V domain is generated through conventional mech­anisms such as V(D)J recombination and somatic hypermutation. In addition, marsupial TCRμ contains the only germline-encoded V gene (Vμj), with pre­joined V, D and J segments discovered in mammals (Miller 2010).

Fig.

Unlike other TCR chains, two isoforms of TCRμ are expressed in marsupials and described in Fig. 7.1 (Miller 2010). TCRμ1.0 contains a single V and C domain simi­lar to conventional TCR, however the V domain is formed by germline encoded Vμj and is thought to be non-viable. This isoform is predominantly expressed in the thymus and its potential functions remain unknown (Miller 2010). The second isoform, TCRμ2.0, has two V domains and a single C domain. The first V domain is formed by conventional V(D)J recombination, and the second domain by Vμj (Miller 2010). Only TCRμ 2.0 is expressed in the platypus; however, unlike in marsupi­als, both V domains are formed by V(D)J recombination (Wang et al. 2011). TCRμ D segments could not be iden­tified in the first version of the platypus genome (Wang et al. 2011) and were not characterised in the latest genome assembly (Zhou et al. 2021). In order to be func­tional, TCRμ presumably forms a heterodimer with a conventional TCR chain. Given the similarities with TCRδ, it is likely that TCRμ pairs with TCRγ. The func­tion of TCRμ+ T lymphocytes remains unknown (Miller 2010).

3.2 Major histocompatibility complex

The MHC is a family of genes essential for innate and adaptive immunity in all jawed vertebrates. As a result it is one of the most gene-rich and polymorphic regions of the genome. Loci for MHC classes I and II are involved in antigen processing and presentation to T lymphocytes and also encode immune molecules such as complement factors and cytokines. Several MHC class I and II gene families are unique to marsupials and monotremes, although orthologs with eutherian genes have been iden­tified (Cheng et al.

Genomic organisation of the eutherian MHC is highly conserved; all three regions are contiguous and ordered class I-III-II within the genome. Detailed characterisa­tion and comparative analysis of the highly contracted black flying-fox MHC-I and -II regions led to the discov­ery of a stepwise duplication process of MHC-I genes within the eutherian class I region (Ng et al. 2016) and the presence of an ancient class II duplication block (Ng et al. 2017), thereby filling an important phylogenetic gap in the evolution of the mammalian MHC region. Bat MHC-I molecules also contain unique peptide-binding grooves, which may increase the efficiency and diversity

Table 7.1. Antibodies that have successfully been used to stain monotreme, marsupial and bat immune tissues and cell populations*

104 CurrentTherapyin MedicineofAustraIian Mammals

¹Rawson etal. 2002; ²OId and Deane 2002; ³Adamski and Demmer 2000; ⁴WiIkinson etal.

*Organised into species-specific antibodies designed for marsupials, monotremes and bats, and human/rodent antibodies that have cross-reactivity.

- Monotreme, marsupial and bat immunology 105

Table 7.2. Monotreme, marsupial and bat cytokines are classified according to their expression by and/or influence on the four major T lymphocyte lineages in eutherian mammals: Th1, Th2, Treg and T 17

N/A represents cytokines which have not been characterised in these species. IFN = interferons, IL = interleukins, LT = lymphotoxin, TGF = transforming growth factor, TNF = tumour necrosis factor, TSLP = thymic stromal lymphopoietin.

¹Wong etal. 2011; ²Alsemgeest etal. 2015; ³Harrison etal. 1999, ⁴Harrison and Deane 1999; ⁵Harrison and Deane 2000; ⁶Young 2011; ⁷Hawken et al. 1999; ⁸Alsemgeest, Old and Young 2013; ⁹Young and Harrison 2010; ¹⁰Suthers, Old and Young 2016; ¹¹Krause and Petska 2005; ¹²Renfree etal. 2011; ¹³Morris etal. 2015; ¹⁴Matthew etal. 2013a, ¹⁵2013b; ¹⁶Morris et al. 2014; ¹⁷Matthew etal. 2014; ¹⁸Young etal. 2012; ¹⁹Wedlock, Aldwell and Buddle 1996; ²⁰Wedlock, Aldwell and Buddle 1998; ²¹Wedlock etal. 1999; ²²Borthwick etal. 2016; ²³Peel etal. 2022; ²⁴Wong et al. 2006; ²⁵Zhou et al. 2016.

of viral antigen presentation, thus conferring their unique ability to control viral infections without overt disease (Ng et al.

Monotreme and marsupial MHC are similar in size and complexity to those of eutherians; however, organisation aligns with that of non-mammals (Belov et al. 2006; Dohm et al. 2007). In most marsupials, class I and II genes are interspersed to create a unique class I-II region, reflecting the organisation of the ancestral mam­malian MHC (Belov et al. 2006; Dohm et al. 2007; Cheng

Table 7.3. Number of characterised class I and II MHC loci in marsupial and monotreme species

¹Belov etal.

et al. 2017; Johnson et al. 2018; Peel et al. 2022). Some class I genes are unlinked to the MHC region and are located on different chromosomes. Monotremes are extreme in that their MHC is located on different pairs of sex chromosomes (Dohm et al. 2007; Zhou et al. 2021).

In wildlife conservation, MHC diversity is used as a measure of immunological fitness within a population because low MHC diversity limits the ability of individuals to respond to emerging infectious diseases. Extremely low MHC class I and II diversity in the Tasma­nian devil facilitated transmission of devil facial tumour disease across Tas. (Cheng et al. 2012a). Recently, MHC class II variants have been associated with chlamydial infection in the koala. Variation in population size and environmental and pathogen pressures (Schad et al. 2012) may have driven the evolution of extreme differences in MHC-II allelic polymorphism among different microchi- ropteran species.

2.5 Immunoglobulins

Immunoglobulins are proteins produced by B lympho­cytes and are essential for the adaptive immune response. Immunoglobulins are formed by two heavy chains (IGH) and two light chains (IGL) joined by disulfide bonds. Both the IGH and IGL contain a constant region (C), which defines the function of the immunoglobulin, and a varia­ble region (V), which binds antigen. Marsupials, mono- tremes and bats utilise four types of IGH constant regions (Cα, Cγ, Cμ and Cε), which define four types of immuno­globulins (IgA, IgG, IgM and IgE, respectively) (Aveskogh et al. 1999; Belov and Hellman 2003; Butler et al. 2011). IgD has not been identified in marsupials, but a homo­logue has been characterised in the platypus (Miller 2010). IgD could not be identified in immune tissue transcrip­tomes of the only Australian bat species studied, the black flying-fox (Papenfuss et al. 2012), although IgD has been identified in insectivorous bat species from the USA (Butler et al. 2011). Similar to its mammalian counter­parts, the black flying-fox has two immunoglobulin light chains, κ and λ (Papenfuss et al. 2012). Monotremes and marsupials also follow this pattern (Belov et al. 2001; Belov et al. 2002; Peel et al. 2022).

Several IgA and IgG isotypes exist in eutherian mam­mals, but only single isotypes of both immunoglobulins are present in marsupials (Morris et al. 2014; Peel et al. 2022). The woylie is the only marsupial genome studied to date which contains three copies of Cγ, all other mar­supials encode only one copy (Peel et al. 2022). Mono- tremes have two highly divergent IgA and IgG isotypes (Belov and Hellman 2003), while bats only have a single IgA isotype but multiple IgG isotypes, which diversified after speciation (Butler et al. 2011).

Antibody diversity is essential for binding a vast array of antigens and for functional adaptive immunity. Diver­sity is generated by variable regions within the IGH and IGL chains. Some marsupials encode a high number of VH genes, up to 147 in the koala (Peel et al. 2022). Despite this, IGH contributes little to overall antibody diversity in monotremes and marsupials, because of the restricted diversity of germline-encoded V_H genes, all of which belong to the ancestral V_h3 family (Aveskogh et al. 1999; Miller 2010). This is compensated by a diverse repertoire of IGL variable regions (V_l), which generate the majority of antibody diversity through V(D)J recombination and somatic hypermutation (Peel et al. 2022). The platypus is the exception, with limited V_L diversity; however, it uses other IGH segments to generate variation (Miller 2010).

Unlike monotremes and marsupials, eutherian mam­mals, including bats, have high IGH diversity, with repre­sentation from all seven V_H families within a species. Bats have an unusually diverse repertoire of V_H genes, which belong to the V_h3 family (Butler et al. 2011). Unlike other eutherian mammals, bats may rely more heavily on the large pool of germline-encoded V_h genes to generate IGH diversity rather than on somatic hypermutation (Butler et al. 2011). To date, there is a lack of research sur­rounding bat immunoglobulins. Studies of serum immu­noglobulin levels discovered abundant IgG and IgM, but extremely low concentrations or total lack of IgA (Wynne et al. 2013). IgA is the main immunoglobulin within mucosal secretions, but in bats high quantities of IgG are present (Wynne et al. 2013).

3.