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Cells expressing variable lymphocyte receptor C ( VLRC ) genes that encode VLRC receptors, which are used by jawless vertebrates to react with antigens, are defined as a second T-cell-like lymphocyte subset (the first being VLRA-bearing cells); distinct properties of these two T-cell-like subsets are reminiscent of the distinction between αβ and γδ T cells in jawed vertebrates. A glimpse of ancient immune systems The jawless vertebrates have an adaptive immune system that uses somatically diversified leucine-rich repeat receptors (VLRs) to recognize antigens, rather than the T- and B-cell receptors of jawed vertebrates. However the VLRB- and VLRA-bearing cells have features that resemble B and T cells, respectively. This study defines a third lineage of lymphocytes in a jawless vertebrate — the lamprey — that expresses the recently described VLRC receptors. The differences between the lamprey's two T-cell-like lymphocyte subsets echo the distinction between the αβ and γδ T-cell lineages of jawed vertebrates, suggesting that functional specialization of distinct T-cell-like lineages was an ancient feature of a primordial immune system. Jawed vertebrates (gnathostomes) and jawless vertebrates (cyclostomes) have different adaptive immune systems 1 , 2 . Gnathostomes use T- and B-cell antigen receptors belonging to the immunoglobulin superfamily 3 , 4 . Cyclostomes, the lampreys and hagfish, instead use leucine-rich repeat proteins to construct variable lymphocyte receptors (VLRs), two types of which, VLRA and VLRB, are reciprocally expressed by lymphocytes resembling gnathostome T and B cells 5 , 6 , 7 . Here we define another lineage of T-cell-like lymphocytes that express the recently identified VLRC receptors 8 , 9 . Both VLRC + and VLRA + lymphocytes express orthologues of genes that gnathostome γδ and αβ T cells use for their differentiation, undergo VLRC and VLRA assembly and repertoire diversification in the ‘thymoid’ gill region, and express their VLRs solely as cell-surface proteins. Our findings suggest that the genetic programmes for two primordial T-cell lineages and a prototypic B-cell lineage were already present in the last common vertebrate ancestor approximately 500 million years ago. We propose that functional specialization of distinct T-cell-like lineages was an ancient feature of a primordial immune system.

As we celebrate 50 years since his seminal Nature paper describing separate lineages for B cells and T cells in the chicken, Max Cooper looks back at the early discoveries that made this breakthrough possible and describes how the B cell field emerged. The separate development of functionally intertwined lineages of lymphocytes known as B cells and T cells is now recognized as a fundamental organizing principle of the adaptive immune system in all vertebrates. Immunologists strive to define the different sublineages of the clonally diverse B cells and T cells, how they interact with each other and how they interact with innate lymphoid cells and other elements of the innate immune system to counter infections, cancer and the development of autoimmune and inflammatory diseases. On the 50th anniversary of the recognition of B cells as a discrete cell lineage, this Timeline article recounts some of the milestones marking the development of the concept that B cells are a functionally and developmentally distinct arm of the adaptive immune system.

Jawless vertebrates use variable lymphocyte receptors (VLR) comprised of leucine-rich-repeat (LRR) segments as counterparts of the immunoglobulin-based receptors that jawed vertebrates use for antigen recognition. Highly diverse VLR genes are somatically assembled by the insertion of variable LRR sequences into incomplete germline VLRA and VLRB genes. Here we show that in sea lampreys ( Petromyzon marinus ) VLRA and VLRB anticipatory receptors are expressed by separate lymphocyte populations by monoallelic VLRA or VLRB assembly, together with expression of cytosine deaminase 1 ( CDA1 ) or 2 ( CDA2 ), respectively. Distinctive gene expression profiles for VLRA + and VLRB + lymphocytes resemble those of mammalian T and B cells. Although both the VLRA and the VLRB cells proliferate in response to antigenic stimulation, only the VLRB lymphocytes bind native antigens and differentiate into VLR antibody-secreting cells. Conversely, VLRA lymphocytes respond preferentially to a classical T-cell mitogen and upregulate the expression of the pro-inflammatory cytokine genes interleukin-17 ( IL-17 ) and macrophage migration inhibitory factor ( MIF ). The finding of T-like and B-like lymphocytes in lampreys offers new insight into the evolution of adaptive immunity. Double immunity in lampreys Lampreys and hagfish, the last of the jawless vertebrates, attract the attention of immunologists as their adaptive immune system rivals that of humans in its flexibility. In humans, each lymphocyte expresses a unique anticipatory receptor for an antigen, constructed from variable, diversity and joining segments. Jawless vertebrates use variable lymphocyte receptors composed of leucine-rich-repeat protein segments with a non-varying stalk tethered to the lymphocyte surface. New work on the sea lamprey reveals yet more parallels with humans: they have a compartmentalized adaptive immune system containing cells that resemble cytokine-secreting T cells and antibody-secreting B cells of the mammalian adaptive immune system. This points to a split in lymphocyte differentiation along T- and B-like cell lineages much earlier in evolution than previously appreciated, such that their system was either a precursor of, or evolved alongside, our own immune system. Variable lymphocyte receptors (VLRs) are used for antigen recognition in jawless vertebrates. Distinctive gene expression profiles for VLRA + and VLRB + lymphocytes in lampreys are now shown to resemble those of mammalian T and B cells, offering insight into the evolution of adaptive immunity.

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. Extant jawless vertebrates, such as lampreys and hagfishes, express variable lymphocyte receptors (VLRs) that are different from adaptive immune receptors in mammals. Here, the authors show that, in addition to the well-described five VLRs, a sixth VLR is expressed in lamprey T-like cells and evolved at least 250 million years ago.

Three types of variable lymphocyte receptor (VLR) genes, VLRA, VLRB, and VLRC, encode antigen recognition receptors in the extant jawless vertebrates, lampreys and hagfish. The somatically diversified repertoires of these VLRs are generated by serial stepwise copying of leucine-rich repeat (LRR) sequences into an incomplete germline VLR gene. Lymphocytes that express VLRA or VLRC are T cell–like, while VLRB-expressing cells are B cell–like. Here, we analyze the composition of the VLRB locus in different jawless vertebrates to elucidate its configuration and evolutionary modification. The incomplete germline VLRB genes of two hagfish species contain short noncoding intervening sequences, whereas germline VLRB genes in six representative lamprey species have much longer intervening sequences that exhibit notable genomic variation. Genomic clusters of potential LRR cassette donors, fragments of which are copied to complete VLRB gene assembly, are identified in Japanese lamprey and sea lamprey. In the sea lamprey, 428 LRR cassettes are located in five clusters spread over a total of 1.7 Mbp of chromosomal DNA. Preferential usage of the different donor cassettes for VLRB assemblage is characterized in our analysis, which reveals evolutionary modifications of the lamprey VLRB genes, elucidates the organization of the complex VLRB locus, and provides a comprehensive catalog of donor VLRB cassettes in sea lamprey and Japanese lamprey.

Variable lymphocyte receptors (VLRs) rather than antibodies play the primary role in recognition of antigens in the adaptive immune system of jawless vertebrates. Combinatorial assembly of leucine-rich repeat (LRR) gene segments achieves the required repertoire for antigen recognition. We have determined a crystal structure for a VLR-antigen complex, VLR RBC36 in complex with the H-antigen trisaccharide from human blood type O erythrocytes, at 1.67 angstrom resolution. RBC36 binds the H-trisaccharide on the concave surface of the LRR modules of the solenoid structure where three key hydrophilic residues, multiple van der Waals interactions, and the highly variable insert of the carboxyl-terminal LRR module determine antigen recognition and specificity. The concave surface assembled from the most highly variable regions of the LRRs, along with diversity in the sequence and length of the highly variable insert, can account for the recognition of diverse antigens by VLRs.

Jawless vertebrates (cyclostomes) have an alternative adaptive immune system in which lymphocytes somatically diversify their variable lymphocyte receptors (VLR) through recombinatorial use of leucine-rich repeat cassettes during VLR gene assembly. Three types of these anticipatory receptors in lampreys (VLRA , VLRB , and VLRC) are expressed by separate lymphocyte lineages. However, only two VLR genes (VLRA and VLRB) have been found in hagfish. Here we have identified a third hagfish VLR , which undergoes somatic assembly to generate sufficient diversity to encode a large repertoire of anticipatory receptors. Sequence analysis, structural comparison, and phylogenetic analysis indicate that the unique hagfish VLR is the counterpart of lamprey VLRA and the previously identified hagfish “VLRA” is the lamprey VLRC counterpart. The demonstration of three orthologous VLR genes in both lampreys and hagfish suggests that this anticipatory receptor system evolved in a common ancestor of the two cyclostome lineages around 480 Mya.

Background/Objectives: Receptor-mediated transcytosis utilizing the native transporters at the blood–brain barrier (BBB) is a growing strategy for the delivery of therapeutics to the brain. One of the major challenges in identifying appropriate human transcytosis targets is that there is a species-specific transporter expression profile at the BBB, complicating translation of successful preclinical candidates into humans. In an effort to overcome this obstacle and identify proteins capable of binding human-relevant BBB ligands, we generated and screened a BBB-targeting library against human-induced pluripotent stem cell-derived brain microvascular endothelial-like cells (iPSC-derived BMEC-like cells). As targeting molecules, we used lamprey antibodies, known as variable lymphocyte receptors (VLRs), and generated a VLR library by immunizing lamprey with iPSC-derived BMEC-like cells, and inserting the resultant VLR repertoire into the yeast surface display system. Methods: The yeast displayed VLR library was then panned against human iPSC-derived BMEC-like cells and lead VLRs were validated using human in vitro models and mouse and human ex vivo brain tissue sections. Results: Finally, brain uptake for a set of VLRs was validated in mice. Of the 15 lead VLR candidates, 14 bound to human BBB antigens, and 10 bound to the murine BBB. Pharmacodynamic testing using the neuroactive peptide neurotensin indicated that the lead candidate, VLR2G, could cross the mouse BBB after intravenous injection and deliver sufficient neurotensin payload to generate a pharmacological response and lower systemic body temperature. Conclusions: Together, these results demonstrate the application of a novel screening technique capable of identifying a VLR with human relevance that can cross the BBB and deliver a payload.

Jawless vertebrates are pivotal representatives for studies of the evolution of adaptive immunity due to their unique position in chordate phylogeny. Lamprey and hagfish, the extant jawless vertebrates, have an alternative lymphocyte-based adaptive immune system that is based on somatically diversifying leucine-rich repeat (LRR)-based antigen receptors, termed variable lymphocyte receptors (VLRs). Lamprey T-like and B-like lymphocyte lineages have been shown to express VLRA and VLRB types of anticipatory receptors, respectively. An additional VLR type, termed VLRC, has recently been identified in arctic lamprey (Lethenteron camtschaticum), and our analysis indicates that VLRC sequences are well conserved in sea lamprey (Petromyzon marinus), L. camtschaticum , and European brook lamprey (Lampetra planeri). Genome sequences of P. marinus were analyzed to determine the organization of the VLRC-encoding locus. In addition to the incomplete germ-line VLRC gene, we have identified 182 flanking donor genomic sequences that could be used to complete the assembly of mature VLRC genes. Donor LRR cassettes were classifiable into five basic structural groups, the composition of which determines their order of use during VLRC assembly by virtue of sequence similarities to the incomplete germ-line gene and to one another. Bidirectional VLRC assembly was predicted by comparisons of mature VLRC genes with the sequences of donor LRR cassettes and verified by analysis of partially assembled intermediates. Biased and repetitive use of certain donor LRR cassettes was demonstrable in mature VLRC s. Our analysis provides insight into the unique molecular strategies used for VLRC gene assembly and repertoire diversification.

Although jawless vertebrates are apparently capable of adaptive immune responses, they have not been found to possess the recombinatorial antigen receptors shared by all jawed vertebrates. Our search for the phylogenetic roots of adaptive immunity in the lamprey has instead identified a new type of variable lymphocyte receptors (VLRs) composed of highly diverse leucine-rich repeats (LRR) sandwiched between amino- and carboxy-terminal LRRs. An invariant stalk region tethers the VLRs to the cell surface by means of a glycosyl-phosphatidyl-inositol anchor. To generate rearranged VLR genes of the diversity necessary for an anticipatory immune system, the single lamprey VLR locus contains a large bank of diverse LRR cassettes, available for insertion into an incomplete germline VLR gene. Individual lymphocytes express a uniquely rearranged VLR gene in monoallelic fashion. Different evolutionary strategies were thus used to generate highly diverse lymphocyte receptors through rearrangement of LRR modules in agnathans (jawless fish) and of immunoglobulin gene segments in gnathostomes (jawed vertebrates).