Martin F. Flajnik

Origin and evolution of the adaptive immune system: genetic events and selective pressures

The adaptive immune system (AIS) in mammals, which is centred on lymphocytes bearing antigen receptors that are generated by somatic recombination, arose approximately 500 million years ago in jawed fish. This intricate defence system consists of many molecules, mechanisms and tissues that are not present in jawless vertebrates. Two macroevolutionary events are believed to have contributed to the genesis of the AIS: the emergence of the recombination-activating gene (RAG) transposon, and two rounds of whole-genome duplication. It has recently been discovered that a non-RAG-based AIS with similarities to the jawed vertebrate AIS — including two lymphoid cell lineages — arose in jawless fish by convergent evolution. We offer insights into the latest advances in this field and speculate on the selective pressures that led to the emergence and maintenance of the AIS.

Comparative analyses of immunoglobulin genes: surprises and portents

The study of immunoglobulin genes in non-mouse and non-human models has shown that different vertebrate groups have evolved distinct methods of generating antibody diversity. By contrast, the development of T cells in the thymus is quite similar in all of the species that have been examined. The three mechanisms by which B cells uniquely modify their immunoglobulin genes — somatic hypermutation, gene conversion and class switching — are increasingly believed to share some fundamental mechanisms, which studies in different vertebrate groups have helped (and will continue to help) to resolve. When these mechanisms are better understood, we should be able to look to the constitutive pathways from which they have evolved and perhaps determine whether the rearrangement of variable, diversity and joining antibody gene segments — V(D)J recombination — was superimposed on an existing adaptive immune system.

The Translesion DNA Polymerase ζ Plays a Major Role in Ig and bcl-6 Somatic Hypermutation

Ig somatic mutations would be introduced by a polymerase (pol) while repairing DNA outside main DNA replication. We show that human B cells constitutively express the translesion pol zeta, which effectively extends DNA past mismatched bases (mispair extender), and pol eta, which bypasses DNA lesions in an error-free fashion. Upon B cell receptor (BCR) engagement and coculture with activated CD4+ T cells, these lymphocytes upregulated pol zeta, downregulated pol eta, and mutated the Ig and bcl-6 genes. Inhibition of the pol zeta REV3 catalytic subunit by specific phosphorothioate-modified oligonucleotides impaired Ig and bcl-6 hypermutation and UV damage-induced DNA mutagenesis, without affecting cell cycle or viability. Thus, pol zeta plays a critical role in Ig and bcl-6 hypermutation, perhaps facilitated by the downregulation of pol eta.

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ‘fossil’ transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17–18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Decreased Frequency of Somatic Hypermutation and Impaired Affinity Maturation but Intact Germinal Center Formation in Mice Expressing Antisense RNA to DNA Polymerase ζ

To examine a role of DNA polymerase ζ in somatic hypermutation, we generated transgenic mice that express antisense RNA to a portion of mouse REV3, the gene encoding this polymerase. These mice express high levels of antisense RNA, significantly reducing the levels of endogenous mouse REV3 transcript. Following immunization to a hapten-protein complex, transgenic mice mounted vigorous Ab responses, accomplished the switch to IgG, and formed numerous germinal centers. However, in most transgenic animals, the generation of high affinity Abs was delayed. In addition, accumulation of somatic mutations in the VH genes of memory B cells from transgenic mice was decreased, particularly among those that generate amino acid replacements that enhance affinity of the B cell receptor to the hapten. These data implicate DNA polymerase ζ, a nonreplicative polymerase, in the process of affinity maturation, possibly through a role in somatic hypermutation, clonal selection, or both.

Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display

The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

Ancestral organization of the MHC revealed in the amphibian Xenopus.

With the advent of the Xenopus tropicalis genome project, we analyzed scaffolds containing MHC genes. On eight scaffolds encompassing 3.65 Mbp, 122 MHC genes were found of which 110 genes were annotated. Expressed sequence tag database screening showed that most of these genes are expressed. In the extended class II and class III regions the genomic organization, excluding several block inversions, is remarkably similar to that of the human MHC. Genes in the human extended class I region are also well conserved in Xenopus, excluding the class I genes themselves. As expected from previous work on the Xenopus MHC, the single classical class I gene is tightly linked to immunoproteasome and transporter genes, defining the true class I region, present in all nonmammalian jawed vertebrates studied to date. Surprisingly, the immunoproteasome gene PSMB10 is found in the class III region rather than in the class I region, likely reflecting the ancestral condition. Xenopus DMα, DMβ, and C2 genes were identified, which are not present or not clearly identifiable in the genomes of any teleosts. Of great interest are novel V-type Ig superfamily (Igsf) genes in the class III region, some of which have inhibitory motifs (ITIM) in their cytoplasmic domains. Our analysis indicates that the vertebrate MHC experienced a vigorous rearrangement in the bony fish and bird lineages, and a translocation and expansion of the class I genes in the mammalian lineage. Thus, the amphibian MHC is the most evolutionary conserved MHC so far analyzed.

Changes in the immune system during metamorphosis of Xenopus

Profound immunological changes occur as tadpoles metamorphose into adult amphibians. These include the expression of a different antibody repertoire, a lessening of skin graft tolerance, the appearance on leukocytes of class I MHC antigens. Here Martin Flajnik and his colleagues review what is known of these changes in Xenopus and speculate on how they may occur.

Which came first, MHC class I or class II?

The topic of this paper, the origins of MHC proteins, seems too difficult to hope to understand. It is our intention, however, to introduce an idea that may influence the way one views the evolution of the MHC. MHC-encoded class I and class II proteins are composed of four extracellular domains, each made up of approximately 90 amino acids. The two membrane-proximal domains are members of the C-1 set of the immunoglobulin (Ig) superfamily (Williams and Barclay 1988). The two two membrane distal domains of class I (and presumably class II) combine to form a peptide-binding cleft composed of a floor of eight /3 strands upon which rest two antiparallel a helices (Bjorkman et al. 1987). The MHC-encoded class I e~ chain is composed of three domains, and is non-covalently associated with the non-MHC encoded /32-microglobulin (Silver and Hood 1974). The peptide-binding domains, both found in the a chain (~-1 and a-2), form an intramolecular dimer (Bjorkman et al. 1987). Both class II proteins are MHCencoded and composed of two domains each; the membrane-distal domains probably form an intermolecular dimer, perhaps stabilized by the peptide (Brown et al. 1988; Mellins et al. 1990). While the membrane-distal domains of MHC molecules are certainly members of the Ig superfamily, the membrane-distal domains that comprise the peptidebinding region are unique and unrelated to any described proteins (Kaufman and Strominger 1982; Bjorkman et al. 1987). Since the peptide-binding domains are approximately the same size as Ig domains, and the intradomain disulfide bond of MHC proteins (in a-2 of class I and/3-1 of class II) and Ig domains is formed by cysteinyl residues spaced approximately the same distance apart, some investigators believe that the peptide-binding domains may be derived from Ig-like domains. In addition, low amino

Expression of MHC Class II Antigens During Xenopus Development

Larval and adult forms of the amphibian Xenopus differ in their MHC class II .expression. In tadpoles, class II epitopes can be detected by monoclonal antibodies only on B cells, macrophages (whatever their location), spleen reticulum, thymus epithelium, and the pharyngobuccal cavity. In contrast, all adult T cells express class II on their surface. The transitions in class II expression occur at metamorphosis and are accompanied by other changes. The skin is invaded by class II positive dendritic cells, and the skin glands differentiate and also express class II. The gut, which expressed class II in discrete areas of the embryonic tissue, becomes invaded with B cells, and its epithelium also becomes class II positive.