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Adaptive immunity is often viewed as a defining innovation of vertebrates, characterized by somatically diversified antigen receptors and clonal lymphocyte lineages. Yet the evolutionary origins of such systems remain incompletely understood. In this review, we examine adaptive immunity from a comparative perspective across Metazoa, focusing on the design principles that link molecular diversification, immune cell differentiation, and proliferative dynamics. We first outline the two adaptive immune architectures found in vertebrates. Jawed vertebrates employ immunoglobulin-based and T-cell receptor-based recognition generated through recombination-activating gene (RAG)-mediated V(D)J recombination, whereas jawless vertebrates assemble variable lymphocyte receptors using cytidine-deaminase-dependent diversification of leucine-rich repeat modules. Despite their distinct molecular entities, these systems converge on shared design principles, including somatic diversification, developmental restriction of genome editing, immune cell differentiation, and specialized microenvironments for immune education. To introduce evolutionarily more ancient systems, several diversification mechanisms of antigen receptors in invertebrates will be subsequently surveyed. These systems generate substantial molecular diversity without canonical clonal selection, suggesting that immune recognition and diversification can be achieved through multiple evolutionary strategies. Particular attention is given to emerging insights into invertebrate immune cell diversification, where single-cell transcriptomics is revealing complex hematopoietic lineages and regulatory programs. These observations suggest that adaptive immunity did not emerge abruptly but rather represents one solution within a broader evolutionary landscape of immune diversification strategies. Understanding how diversification, proliferation, and cellular organization interact across animal lineages will help clarify the fundamental design constraints that shaped the evolution of vertebrate adaptive immune systems.

All vertebrate adaptive immune systems exhibit distinct lymphocyte lineages. In jawless vertebrates, such as lampreys, T-like cells are thought to develop in lympho-epithelial structures at the tips of gill filaments, termed thymoids. However, it is unclear whether thymoids are functionally equivalent to the thymus of jawed vertebrates. Indeed, because the structural modules that are somatically assembled to form the antigen receptors of jawless and jawed vertebrates differ, development and selection of T cells may be governed by clade-specific genetic networks. To address this question, we have replaced the mouse Foxn1 gene, a key regulator of the thymic microenvironment in jawed vertebrates, with the orthologous FOXN1 lamprey gene, which is expressed in the thymoids alongside genes orthologous to known targets of the mouse Foxn1 transcription factor. The reconstituted thymi support normal T cell development, and, to a lesser extent, also support B cell development, indicating that the lamprey FOXN1 gene can rescue the block of thymic epithelial cell differentiation in mice deficient for the endogenous Foxn1 gene. The absence of overt autoimmunity in transgenic mice suggests that the reconstituted thymic microenvironment directs the development of a self-tolerant T cell repertoire. These findings highlight the remarkable similarity of thymic epithelial functions in jawed and jawless vertebrates, despite more than 500 My of independent evolution. Our results thus suggest that the emergence of the Foxn1 transcription factor in the common ancestor of vertebrates was associated with the advent of a specialized tissue environment supporting the development and selection of T cells.

All extant jawless vertebrates (lampreys and hagfishes) possess a unique adaptive immune system characterized by highly variable lymphocyte receptors (VLR) that are assembled in developing lymphocytes using leucine-rich-repeat donor cassettes. Five VLR types have been identified in lampreys: VLRA, VLRB, VLRC, VLRD, and VLRE. VLRB-expressing lymphocytes are functional analogs to B cells, whereas VLRA, VLRC, VLRD, and VLRE-expressing lymphocytes are more akin to T cells of jawed vertebrates. Here we define an additional VLR, designated VLRF. VLRF is phylogenetically closest to VLRA, with which it likely shares a common ancestral gene of at least 250 million years in the past. VLR assembly analyses show that VLRA, VLRC, VLRD, VLRE, and VLRF share donor cassettes through long-range intra- and inter-chromosomal interactions, whereas VLRB utilizes a distinct, dedicated cassette set. The pattern of gene expression, donor cassette usage, and distinctive amino acid composition in the C-terminal stalk suggest that VLRF⁺ lymphocytes may represent an additional T-like sub-lineage, adding further complexity to the VLR-based adaptive immune system.

Adaptive immunity of the type found in mammals is phylogenetically restricted to the jawed vertebrates; however, a second form of somatically diversifying receptors is present in the living jawless vertebrates, the lampreys and hagfishes. Unlike the immunoglobulin domain-based antibodies and T cell receptors of jawed vertebrates, the variable lymphocyte receptors (VLRs) of jawless vertebrates are based on leucine-rich repeat (LRR) proteins. One type of these, variable lymphocyte receptor B (VLRB), is expressed as both a cell surface receptor on B-like cells and as a soluble polymeric antibody, analogous to the immunoglobulins of jawed vertebrates. The genomic locus encoding VLRB consists of an incomplete germline gene with two regions that make up the invariant ends of the mature receptor separated by a noncoding intervening region. The germline VLRB gene is surrounded by hundreds of donor cassettes encoding LRR fragments that contribute to the variable portion of the mature receptor by incorporation into the locus in a process of stepwise somatic assembly. VLRB is expressed from a single allele as clonal receptors on B cell-like lymphocytes and secreted as antibody from plasma-like cells. Here we discuss the structure of the VLRB monomer and antibody, the genomic organisation of the complex VLRB locus in lamprey, the B-like lymphocytes that express VLRB, the role of cytidine deaminases in the assembly of mature VLRB and the applications of these lamprey antibodies in biomedicine.

Jawless vertebrates possess an alternative adaptive immune system in which antigens are recognized by variable lymphocyte receptors (VLRs) generated by combinatorial assembly of leucine-rich repeat (LRR) cassettes. Three types of receptors, VLRA, VLRB, and VLRC, have been previously identified. VLRA- and VLRC-expressing cells are T cell-like, whereas VLRB-expressing cells are B cell-like. Here, we report two types of VLRs in lampreys, VLRD and VLRE, phylogenetically related to VLRA and VLRC. The germline VLRD and VLRE genes are flanked by 39 LRR cassettes used in the assembly of mature VLRD and VLRE, with cassettes from chromosomes containing the VLRA and VLRC genes also contributing to VLRD and VLRE assemblies. VLRD and VLRE transcription is highest in the triple-negative (VLRA−/VLRB−/VLRC−) population of lymphocytes, albeit also detectable in VLRA+ and VLRC+ populations. Tissue distribution studies suggest that lamprey VLRD+ and VLRE+ lymphocytes comprise T-like sublineages of cells.

Genetic conflicts shape the genomes of prokaryotic and eukaryotic organisms. Here, we argue that some of the key evolutionary novelties of adaptive immune systems of vertebrates are descendants of prokaryotic toxin-antitoxin (TA) systems. Cytidine deaminases and RAG recombinase have evolved from genotoxic enzymes to programmable editors of host genomes, supporting the astounding discriminatory capability of variable lymphocyte receptors of jawless vertebrates, as well as immunoglobulins and T cell receptors of jawed vertebrates. The evolutionarily recent lymphoid lineage is uniquely sensitive to mutations of the DNA maintenance methylase, which is an orphaned distant relative of prokaryotic restriction-modification systems. We discuss how the emergence of adaptive immunity gave rise to higher order genetic conflicts between genetic parasites and their vertebrate host.

In vertebrates, the development of lymphocytes from undifferentiated haematopoietic precursors takes place in so-called primary lymphoid organs, such as the thymus. Therein, lymphocytes undergo a complex differentiation and selection process that culminates in the generation of a pool of mature T cells that collectively express a self-tolerant repertoire of somatically diversified antigen receptors. Throughout this entire process, the microenvironment of the thymus in large parts dictates the sequence and outcome of the lymphopoietic activity. In vertebrates, direct genetic evidence in some species and circumstantial evidence in others suggest that the formation of a functional thymic microenvironment is controlled by members of the Foxn1/4 family of transcription factors. In teleost fishes, both Foxn1 and Foxn4 contribute to thymopoietic activity, whereas Foxn1 is both necessary and sufficient in the mammalian thymus. The evolutionary history of Foxn1/4 genes suggests that an ancient Foxn4 gene lineage gave rise to the Foxn1 genes in early vertebrates, raising the question of the thymopoietic capacity of the ancestor common to all vertebrates. Recent attempts to reconstruct the early events in the evolution of thymopoietic tissues by replacement of the mouse Foxn1 gene by Foxn1-like genes isolated from various chordate species suggest a plausible scenario. It appears that the primordial thymus was a bi-potent lymphoid organ, supporting both B cell and T cell development; however, during the course of vertebrate, evolution B cell development was gradually diminished converting the thymus into a site specialized in T cell development.

The adaptive immune systems of all vertebrates rely on self-DNA mutating enzymes to assemble their antigen receptors in lymphocytes of their two principal lineages. In jawed vertebrates, the RAG1/2 recombinase directs V(D)J recombination of B cell and T cell receptor genes, whereas the activation-induced cytidine deaminase AID engages in their secondary modification. The recombination activating genes (RAG) 1 and 2 evolved from an ancient transposon-encoded genome modifier into a self-DNA mutator serving adaptive immunity; this was possible as a result of domestication, involving several changes in RAG1 and RAG2 proteins suppressing transposition and instead facilitating-coupled cleavage and recombination. By contrast, recent evidence supports the notion that the antigen receptors of T-like and B-like cells of jawless vertebrates, designated variable lymphocyte receptors (VLRs), are somatically assembled through a process akin to gene conversion that is believed to be dependent on the activities of distant relatives of AID, the cytidine deaminases CDA1 and CDA2, respectively. It appears, therefore, that the precursors of AID and CDAs underwent a domestication process that changed their target range from foreign nucleic acids to self-DNA; this multi-step evolutionary process ensured that the threat to host genome integrity was minimized. Here, we review recent findings illuminating the evolutionary steps associated with the domestication of the two groups of genome editors, RAG1/2 and cytidine deaminases, indicating how they became the driving forces underlying the emergence of vertebrate adaptive immune systems.

The antibodies of jawless vertebrates consist of leucine-rich repeat arrays encoded by somatically assembled VLRB genes. It is unknown how the incomplete germline VLRB loci are converted into functional antibody genes during B lymphocyte development in lampreys. In Lampetra planeri larvae lacking the cytidine deaminase CDA2 gene, VLRB assembly fails, whereas the T lineage–associated VLRA and VLRC antigen receptor gene assemblies occur normally. Thus, CDA2 acts in a B cell lineage–specific fashion to support the somatic diversification of VLRB antibody genes. CDA2 is closely related to activation-induced cytidine deaminase (AID), which is essential for the elaboration of immunoglobulin gene repertoires in jawed vertebrates. Our results thus identify a convergent mechanism of antigen receptor gene assembly and diversification that independently evolved in the two sister branches of vertebrates.

The onset of lymphocyte development in the vertebrate primordial thymus, about 500 million years ago, represents one of the foundational events of the emerging adaptive immune system. Here, we retrace the evolutionary trajectory of thymopoiesis, from early vertebrates to mammals, guided by members of the Foxn1/4 transcription factor gene family, which direct the differentiation of the thymic microenvironment. Molecular engineering in transgenic mice recapitulated a gene duplication event, exon replacements, and altered expression patterns. These changes predictably modified the lymphopoietic characteristics of the thymus, identifying molecular features contributing to conversion of a primordial bipotent lymphoid organ to a tissue specializing in T cell development. The phylogenetic reconstruction associates increasing efficiency of T cell generation with diminishing B cell-generating capacity of the thymus during jawed vertebrate evolution.